Modified spider silk fiber and use thereof

ABSTRACT

Derivatized major ampullate spidroin protein (MaSp)-based fiber are disclosed. Compositions and/or composites comprising the derivatized fibers and method for producing thereof are further disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Bypass Continuation of PCT Patent Application No. PCT/IL2021/050475 having International filing date of Apr. 25, 2021, which claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application Nos. 63/014,444, filed Apr. 23, 2020, entitled “ANTI-MICROBIAL COMPOSITIONS”, 63/048,135, filed Jul. 5, 2020, entitled “COSMETIC COMPOSITIONS COMPRISING DRAGLINE SPIDER SILK”, 63/144,089, filed Feb. 1, 2021 entitled “COSMETIC COMPOSITIONS COMPRISING DRAGLINE SPIDER SILK”, 63/079,621 filed Sep. 17, 2020, entitled “MODIFIED SPIDER SILK FIBER AND USE THEREOF”, 63/134,343 filed Jan. 6, 2021 entitled “EXTRUDATE COMPOSITIONS COMPRISING DRAGLINE SPIDER SILK” the contents of which are all incorporated herein by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (06939-P0008B-SVX-P-010-US-SQL.xml; Size: 15,491 bytes; and Date of Creation: Oct. 24, 2022) is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention, in some embodiments thereof, is directed to compositions comprising proteins derived from a MaSp (major ampullate spidroin) protein, and the preparation of same and use as cosmeceutical or anti-microbial materials.

BACKGROUND OF THE INVENTION

Dragline spider silk is known in the art as the silk used by the orb-web weaving spiders to construct the frame and radii of their webs as well a lifeline when they fall or escape danger. To be able to perform these tasks, the dragline fiber displays a remarkably high toughness due to combination of high elasticity and strength, which places it as the toughest fiber, whether natural or man-made. For instance, dragline is six times as strong as high-tensile steel in its diameter and three times tougher than Kevlar that is one of the strongest synthetic fibers ever made.

Dragline silk consists of two main polypeptides, mostly referred to as major ampullate spidroin (MaSp) 1 and 2, and also to ADF-3 and ADF-4 in Araneus diadematus. These proteins have apparent molecular masses in the range of 200-720 kDa, depending on sample age and conditions of analysis. The known dragline silk spidroins are composed of highly iterated blocks of alternating alanine-rich segments, forming crystalline P-sheets in the fiber, and glycine-rich segments which are more flexible and mainly lack ordered structure. The C-terminal region is non-repetitive, highly conserved between species, and adopts a-helical conformation. The N-terminal region of dragline silk proteins was also found to be highly conserved between different spidroins, and also between different spider species.

SUMMARY OF THE INVENTION

According to one aspect, there is provided a derivatized porous major ampullate spidroin protein (MaSp)-based fiber, wherein the derivatized porous MaSp-based fiber is characterized by a BET surface area of at least 10 m²/g; the derivatized porous MaSp-based fiber comprises a functional moiety covalently bound to a tyrosine of the porous MaSp-based fiber; the functional moiety comprises any one of: amino, carboxy, nitro, sulfonate, carbonyl, ester, anhydride, carbonate ester, carbamate, cyano, hydroxy, a polymer, or any combination thereof.

In one embodiment, a loading of the functional moiety within derivatized porous MaSp-based fiber is between 0.01 μmol/g and 10 mmol/g.

In one embodiment, functional moiety is covalently bound to a side chain of the tyrosine via a diazo bond, a silyl group, or any combination thereof.

In one embodiment, the polymer is covalently bound to a chelating agent, an antimicrobial agent or any combination thereof.

In one embodiment, chelating agent comprises (i) metal chelating group capable of binding a metal or a salt thereof, (ii) a metal oxide chelating group, or both (i) and (ii).

In one embodiment, metal chelating group comprises iminodiacetate (IDA), DOTA, NOTA, NODA, EDTA, HBED-CC, including any salt, a derivative, or a combination thereof.

In one embodiment, metal oxide chelating group is selected from salicylic acid, phosphonic acid, hydroxamic acid, malonic acid, pyrogallol, and 5-hydroxy-1,4-naphtoquinone, including any salt, or any combination thereof.

In one embodiment, the polymer is selected from the group consisting of polyglutaraldehyde (PGA), polyvinyl alcohol (PVA), polyacrylate (PAA), polyethyleneimine (PEI), polyacrylamide (PAAm), polylysine, polyaniline, polyurethane, polyamide, polyvinyl chloride, silicon crosspolymer, polyvinyl pyrrolidone or any combination thereof.

In one embodiment, a w/w ratio of the polymer to the porous MaSp-based fiber is between 0.001 and 5.

In one embodiment, the functional moiety is further bound to a dye or a pigment.

In one embodiment, the MaSp-based fiber is characterized by a degradation temperature (Td) between 280° C. and 350° C. as determined by differential scanning calorimetry (DSC), and by a glass transition temperature (Tg) between 200° C. and 250° C., as determined by DSC.

In one embodiment, the MaSp-based fiber comprises a repetitive region comprising an amino acid sequence as set forth in Formula 10: ((X₁)_(z)X₂GPGGYGPX₃X₄X₅GPX₆GX₇GGX₈GPGGPGX₉X₁₀; wherein X₁ is, independently, at each instance A or G, Z is an integer between 5 to 30, X₂ is S or G; X₃ is G or E; X₄ is G, S or N; X₅ is Q or Y; X₆ is G or S; X₇ is P or R; X₈ is Y or Q; X₉ is G or S; and X₁₀ is S or G.

In another aspect, there is provided a composite comprising the derivatized porous MaSp-based fiber of the invention bound to any one of a metal, a salt thereof, and a metal oxide particle or any combination thereof.

In one embodiment, the metal, a salt thereof, or the metal oxide particle is bound to the derivatized porous MaSp-based fiber via a chelating agent.

In one embodiment, the chelating agent comprises (i) metal chelating group capable of binding a metal or a salt thereof, (ii) a metal oxide chelating group, or both (i) and (ii).

In one embodiment, the metal chelating group comprises iminodiacetate (IDA), DOTA, NOTA, NODA, EDTA, HBED-CC, including any salt, a derivative, or a combination thereof, and wherein the metal oxide chelating group is selected from salicylic acid, phosphonic acid, hydroxamic acid, malonic acid, pyrogallol, and 5-hydroxy-1,4-naphtoquinone, including any salt, or any combination thereof.

In one embodiment, the molar ratio of the chelating agent to the derivatized porous MaSp-based fiber is between 0.01 and 1.

In one embodiment, the metal oxide particle is selected from titania, zirconia, silica or any combination thereof. In one embodiment, the metal oxide particle is characterized by a particle size between 10 and 5,000 nm.

In one embodiment, the w/w ratio of the derivatized porous MaSp-based fiber to the metal oxide particle within the composite is between 0.01 and 100.

In another aspect, there is provided a method of synthesizing the derivatized porous MaSp-based fiber of the invention, the method comprises reacting the porous MaSp-based fiber with a reagent represented by Formula I:

or by Formula II:

wherein R comprises the functional moiety of the invention; A is selected from aryl, heteroaryl, and alkyl substituted or non-substituted, including any combination thereof, each R1 independently comprises any one of hydrogen, an alkyl, hydroxy or alkoxy, including any combination thereof, a wavy bond represents a linker or a bond; and wherein the reacting comprises conditions sufficient for covalent attachment of the functional moiety to the tyrosine of the porous MaSp-based fiber, thereby obtaining the derivatized porous MaSp-based fiber.

In one embodiment, the covalent attachment is via a diazo bond or via a C—Si.

In one embodiment, R1 comprises —O—C1-10 alkyl.

In one embodiment, A comprises a substituted or non-substituted phenyl.

In one embodiment, the linker comprises a substituted or non-substituted C1-10 alkyl.

In one embodiment, the method further comprises reacting the functional moiety with a polymer having reactivity to the functional moiety, thereby covalently binding the polymer to said porous MaSp-based fiber; wherein said functional moiety comprises an amine or a carboxy group.

In one embodiment, the method further comprises reacting said polymer with a chelating agent having reactivity to said polymer, thereby obtaining said chelating agent covalently bound to the polymer.

In one embodiment, the polymer comprises PGA.

According to another aspect, there is provided a composition comprising a major ampullate spidroin protein (MaSp)-based fiber comprising a microbial interacting protein.

In some embodiments, the microbial interacting protein is a virus interacting protein. In some embodiments, the virus interacting protein is a virus binding receptor. In some embodiments, the virus is a coronavirus.

In some embodiments, the composition further comprises at least one anti-microbial agent. In some embodiments, the anti-microbial agent is selected from the group consisting of: a source of reactive oxygen species (ROS), a carboxylic acid, a quaternary amine, a disinfecting agent, a transition metal, an electrophilic reactive group, an oxidizing agent, an antimicrobial polymer or any combination thereof.

In some embodiments, the anti-microbial agent is bound to the MaSp-based fiber, by a hydrogen bond, Van der Waals bond, or both. In some embodiments, the MaSp-based fiber is coated with a polymeric layer, and wherein the polymeric layer is further bound to the transition metal. In some embodiments, the anti-microbial agent is covalently bound to the MaSp-based fiber. In some embodiments, covalently bound is via a linker comprising a diazo group. In some embodiments, the MaSp-based fiber is covalently bound to a polymer comprising a chelating moiety. In some embodiments, the polymer comprises poly-glutaraldehyde, and wherein the chelating moiety comprises iminodiacetate.

In some embodiments, the composition comprises a transition metal bound to the chelating moiety. In some embodiments, the transition metal is further in contact with an additional transition metal.

According to another aspect, there is provided a composition comprising a major ampullate spidroin protein (MaSp)-based fiber modified with any one of an anti-microbial agent, and a transition metal. In some embodiments, the anti-microbial agent is selected from the group consisting of: a source of reactive oxygen species (ROS), a carboxylic acid, a quaternary amine, a disinfecting agent, a transition metal, an electrophilic reactive group, an oxidizing agent, an antimicrobial polymer or any combination thereof.

In some embodiments, modified comprises covalently bound, non-covalently bound or both.

In some embodiments, the MaSp-based fiber is coated with a polymeric layer, and wherein the polymeric layer is further bound to the transition metal. In some embodiments, covalently bound is via a linker comprising a diazo group. In some embodiments, the MaSp-based fiber is covalently bound to a polymer comprising a chelating moiety.

In some embodiments, the composition comprises a transition metal bound to the chelating moiety. In some embodiments, the transition metal is further in contact with an additional transition metal. In some embodiments, the polymer comprises poly-glutaraldehyde, and wherein the chelating moiety comprises iminodiacetate.

In some embodiments, the composition or the MaSp-based fiber of the invention is characterized by a porosity suitable for capturing a microbe in a range between 50 and 500 nm. In some embodiments, the porosity comprises a median pore size in a range between 1 and 500 nanometers.

In some embodiments, the ratio between the MaSp-based fiber and the anti-microbial agent is 0.01:1 to 1:1.

In some embodiments, the composition is for use in providing an anti-microbial effect to any one of a cream, textile, and substrate.

In some embodiments, the composition comprises 0.01% to 50% (w/w) of the MaSp-based fiber and an additional polymer.

In some embodiments, the additional polymer is selected form the group consisting of thermoplastic polymer, a thermoset an epoxy, a polyester a polyamide, a polyol, a polyurethane, polyethylene, Nylon, a polyacrylate, a polycarbonate, polylactic acid (PLA) or a copolymer thereof a silicon, a liquid crystal polymer, a maleic anhydride grafted polypropylene, polycaprolactone (PCL), rubber, cellulose, or any combination thereof.

According to another aspect, there is provided an article comprising the composition described herein. In some embodiments, the article further comprises 0.01% to 50% w/w of an additional polymer.

In some embodiments, the article is in a form of a fibrous mate, a polymeric layer, or a coating. In some embodiments, the article is characterized by at least one improved anti-microbial property as compared to the property for the article free of the composition.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 represents a multi-layer structure and stepwise preparation process of MsSp-based fibers (termed herein SVXE) coated with Cu.

FIG. 2 represents an image of a nylon fiber enriched (10% w/w) by SVXE coated with Cu.

FIGS. 3A-B represent micrographs of metal-coated SVXE fibers. FIG. 3A: a micrograph of Pd-doped SVXE fiber. FIG. 3B: a micrograph showing a Cu layer deposited on top of Pd-doped SVXE fiber (Cu-SVXE).

FIG. 4 represents an image showing antibacterial properties of Nylon fibers enriched by 10% w/w of Cu-SVXE, versus pristine Nylon fibers. ZOI (zone of inhibition) indicates area free of bacterial growth.

FIG. 5 is a graph representing an IR spectrum of a carboxylated MaSp based fiber (derivatized with aminobenzoic acid via a diazo bond) and an IR spectrum of the pristine MaSp based fiber. * is assigned to a peak corresponding to O—H vibration of the carboxy group. The IR spectrum of the carboxylated MaSp based fiber, represents intensity enhancement of the corresponding peak compared to the pristine MaSp-based fiber, supporting efficient carboxylation of the MaSp based fiber. ** is assigned to a peak corresponding to C—H vibrations of the aromatic hydrogen of tyrosine side chain. The IR spectrum of the carboxylated MaSp based fiber, represents a significant decrease of the peak intensity compared to the pristine MaSp-based fiber, supporting efficient tyrosine diazotization of the carboxylated MaSp based fiber.

FIG. 6 is a graph representing and zeta potential of an aminated MaSp based fiber (Amine), carboxylated MaSp based fiber (Acid), PAA modified aminated MaSp based fiber (PAA), PEI modified carboxylated MaSp based fiber (PEI), and pristine MaSp based fiber (SVXE).

FIGS. 7A-B are SEM images of porous MaSp-based fibers conjugated with polyacrylate (FIG. 7A) or with polyglutaraldehyde (FIG. 7B).

FIG. 8 is a graph presenting differential scanning calorimetry (DSC) curves of the SVX-E at a temperature increase of 25° C.-280° C. (curve 1), cooling back to 50° C. (curve 2), and new increase to 350° C. (curve 3).

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in some embodiments thereof is related to chemically modified, or derivatized MaSp-based fibers. As demonstrated herein below, the inventors successfully synthesized conjugates of the derivatized MaSp-based fiber (e.g. comprising functional moieties bound to the tyrosine of the MaSp-based fiber via a diazo bond or via a silyl group, as described hereinbelow) with various polymers such as PGA, PAA, PVA, PEI, PAAm or a combination thereof (such as PGA-co-PEI). Exemplary non-limiting derivatized MaSp-based fibers, such as amino or PEI modified MaSp-based fibers bearing cationic groups, exhibited positive zeta potential. Surprisingly, MaSp-based fibers characterized by positive zeta potential, exhibited superior adherence to hair (e.g. damaged hair) and to hydrophobic surfaces, such as glass surface.

Furthermore, the inventors utilized some of the derivatized MaSp-based fibers of the invention for complexing metals (such as elemental Cu), so as to obtain MaSp-based fibers characterized by superior electrical conductivity.

According to one aspect, the present invention provides a derivatized porous major ampullate spidroin protein (MaSp)-based fiber, wherein the derivatized porous MaSp-based fiber comprises a functional moiety covalently bound to an amino acid of the porous MaSp-based fiber. In some embodiments, the porous MaSp-based fiber is characterized by a BET surface area of at least 10 m²/g. In some embodiments, the porous MaSp-based fiber is as described hereinbelow. In some embodiments, the derivatized porous MaSp-based fiber comprises a functional moiety covalently bound to a side chain of an amino acid of the porous MaSp-based fiber, and the porous MaSp-based fiber is characterized by a BET surface area of at least 10 m²/g.

In some embodiments, the derivatized porous MaSp-based fiber comprises a functional moiety covalently bound to a side chain of an amino acid of the porous MaSp-based fiber, and the porous MaSp-based fiber is characterized by at least one of: (i) a BET surface area of at least 10 m²/g; (ii) a degradation temperature of between 280 and 350° C.; (iii) glass transition temperature of between 200 and 250° C., and (iv) an amino acid sequence comprising a repetitive region, wherein each repetitive region comprises, independently, an amino acid sequence as set forth in Formula 10 below, or a combination of (i), (ii), (iii) and (iv).

In some embodiments, the derivatized porous MaSp-based fiber comprises a functional moiety covalently bound to a side chain of an amino acid of the porous MaSp-based fiber, wherein the porous MaSp-based fiber comprises a repetitive region, wherein each repetitive region comprises, independently, an amino acid sequence as set forth in Formula 10 below and the derivatized porous MaSp-based fiber is characterized by at least one of: (i) a BET surface area of at least 10 m²/g; (ii) a degradation temperature of between 280 and 350° C.; (iii) glass transition temperature of between 200 and 250° C., or a combination of (i), (ii) and (iii). In some embodiments, the term “porous MaSp-based fiber” and the term “MaSp-based fiber” are used herein interchangeably.

In some embodiments, the functional moiety is covalently bound to an amino acid of the porous MaSp-based fiber. In some embodiments, the functional moiety is covalently bound to an amino acid, wherein the amino acid is selected from tyrosine, serine, cysteine and lysine, threonine, histidine, arginine, aspartic acid and glutamic acid or any combination thereof. In some embodiments, the functional moiety is covalently bound to a side chain of an amino acid, wherein the amino acid is selected from tyrosine, serine, cysteine and lysine, threonine, histidine, arginine, aspartic acid and glutamic acid or any combination thereof. In some embodiments, the functional moiety is covalently bound to a side chain of any of tyrosine, serine, cysteine and lysine or any combination thereof. In some embodiments, the functional moiety is covalently bound to a side chain (e.g. phenol ring) of tyrosine.

In some embodiments, the derivatized porous MaSp-based fiber of the invention comprises a functional moiety covalently bound to a side chain of at least one tyrosine via a diazo bond, a silyl group or both. In some embodiments, the derivatized porous MaSp-based fiber of the invention comprises a diazotized tyrosine, a silylated tyrosine or both. In some embodiments, between 1 and 99%, between 10 and 99%, between 10 and 90%, between 10 and 80%, between 10 and 70%, between 10 and 60%, including any range or value therebetween of the tyrosine residues (or side chains) within the derivatized porous MaSp-based fiber of the invention, are diazotized and/or silylated.

In some embodiments, the derivatized porous MaSp-based fiber of the invention comprises a functional moiety covalently bound to a side chain of at least one tyrosine via a diazo bond, also referred to herein as “diazotized tyrosine”. In some embodiments, the diazotized tyrosine comprises the functional moiety covalently bound to the phenyl ring of the tyrosine via a diazo bond:

In some embodiments, the functional moiety is covalently bound to the porous MaSp-based fiber via any one of diazo, silyl, carbonyl, amide, ester, maleimide, or any combination thereof.

In some embodiments, the functional moiety is covalently bound to at least one tyrosine residue of the porous MaSp-based fiber. In some embodiments, the functional moiety is covalently bound to at least one tyrosine residue via a bond selected from diazo, silyl, ester, carbamate, carbonyl, (O- or S)-thiocarbamate, or any combination thereof. In some embodiments, the functional moiety is covalently bound to at least one tyrosine residue via a diazo bond. In some embodiments, the functional moiety is covalently bound to at least one tyrosine residue via a linker comprising any reactive group capable of forming (i) a diazo bond with the phenol ring of tyrosine (e.g. alkyl diazonium or aryldiazonium group), and/or (ii) a Si—C with the phenol ring of tyrosine (e.g. alkoxysilyl, or halosilyl).

In some embodiments, the composition is substantially devoid of functional moieties and/or polymers adsorbed to the MaSp-based fiber.

In some embodiments, the derivatized porous MaSp-based fiber of the invention comprising the functional moiety covalently bound to the MaSp-based fiber via a diazo bond is represented by Formula 1:

wherein

represents the MaSp-based fiber, A is selected from aryl (e.g. phenyl, or a bicyclic/fused aromatic ring, such as naphthalene), heteroaryl (e.g. C5-6 aromatic ring comprising 1, 2, 3, or 4 heteroatoms selected from O, N and S), and alkyl (e.g. C1-C10 linear or branched alkyl) substituted or non-substituted; wherein R is or comprises the functional moiety of the invention, and wherein a wavy bond represents a linker (or spacer), as described herein.

In some embodiments, the derivatized porous MaSp-based fiber of the invention comprising the functional moiety covalently bound to the MaSp-based fiber via a silyl group is represented by Formula 2:

wherein a wavy bond and R are as described herein, and each R₁ independently comprises any one of hydrogen, an optionally substituted alkyl (e.g. C1-C10 linear or branched alkyl), hydroxy or alkoxy (e.g. C1-C10 alkoxy).

In some embodiments, the functional moiety of the invention comprises any one of a hydroxy group, a mercapto group, an amino group, a carboxylate group, nitro, sulfonate, carbonyl, anhydride, carbonate ester, carbamate, a nitrile group, and a polymer or any combination thereof. In some embodiments, the functional moiety comprises a hydroxy group, a mercapto group, an amino group, a carboxylate group, and a polymer or any combination thereof. In some embodiments, the functional moiety is covalently bound an amino acid of the porous MaSp-based fiber via linear or a branched linker. In some embodiments, each derivatized amino acid of the porous MaSp-based fiber comprises one or more functional moieties covalently bound thereto.

In some embodiments, the functional moiety of the invention is attached to a tyrosine residue via a diazoaryl. In some embodiments, the functional moiety of the invention is bound to the diazoaryl via a linker. In some embodiments, the term “linker” and the term “spacer” are used herein interchangeably.

In some embodiments, the functional moiety (or R) comprises an electrophilic and/or a nucleophilic group. Numerous electrophiles and nucleophiles are well-known in the art.

Non-limiting examples of electrophilic reactive groups include but are not limited to aldehyde, ketone, carboxy, ester, imine, oxime, acyl halide, an active ester (e.g. N-hydroxy succinimide), a chloroformate, an anhydride, an epoxide, an isocyanate, nitro, sulfonate, trialkylammonium, and halo including any alkyl and/or aryl derivative (such as carboxyalkyl, carboxyaryl, alkyl carbonyl, haloalkyl, haloaryl etc.) or any combination thereof.

Non-limiting examples of nucleophilic reactive groups include but are not limited to hydroxy, a mercapto, an amino, phosphine or any combination thereof.

In some embodiments, the functional moiety comprises a plurality of electrophilic and/or a nucleophilic groups. In some embodiments, the functional moiety comprises a polyamine (e.g. a linear polyamine such as spermine or spermidine; or a branched polyamine such as tris (2-aminoethyl) amine). In some embodiments, the functional moiety comprises a polyol (e.g. pentaerythritol, xylitol). In some embodiments, the functional moiety comprises di-, or tri-carboxylic acid (e.g. citric acid, malic acid, succinic acid).

In some embodiments, the functional moiety (or R) comprises any one of alkyl (e.g. C1-10 alkyl linear or branched, such as methyl, ethyl, propyl, butyl, isobutyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl), C1-10 alkoxy (e.g. methoxy, ethoxy, propoxy, ocyloxy), a vinyl group (e.g. vinyloxy), C1-10 alkylamino (e.g. methyl amino, ethyl amino, propyl amino, butyl amino, pentyl amino, hexylamino), protected amine (e.g. by an amine protecting group selected from Fmoc, Boc, Benzyl, CBz, etc.), hydroxy (optionally protected by a hydroxy protecting group, such as tert-butyl, or trimethylsilyl), mercapto (optionally protected by a thiol protecting group), silyl, siloxane (e.g. trialkoxysilane), nitro, sulfonate, cyano, halo, trialkylammonium, aldehyde, ketone, carboxy, ester, imine, oxime, acyl halide, an active ester (e.g. N-hydroxy succinimide), a chloroformate, an anhydride, an epoxide, an isocyanate or any combination thereof.

In some embodiments, the linker (or spacer) comprises an alkyl optionally substituted by any one of carboxy, halo, hydroxy, amino, cycloalkyl, alkyl, nitro, sulfonate, cyano or any combination or derivative thereof. In some embodiments, the linker comprises optionally substituted alkoxy, thioalkyl, aminoalkyl, glycol or any combination thereof. In some embodiments, alkyl including any derivative thereof is as described hereinbelow. In some embodiments, the linker comprises a di-substituted alkyl (e.g. aminohexanoic acid), or a di-substituted heteroalkyl group. In some embodiments, the linker comprises C1-C10 alkyl, a C1-C10 aminoalkyl, a C1-C10 alkoxy, a C1-C10 mercaptoalkyl, a carbonyl derivative (e.g., —C(O)NH—, —C(O)O—, —C(O)—, —C(O)S—, —C(NH)NH—, —C(NH)O—, —C(NH)S—), and optionally comprises one or more heteroatoms (e.g. S, N, O) within the backbone of the linker, or any combination thereof.

In some embodiments, the linker (or spacer) comprises natural and/or unnatural amino acid, alkyl, an amide bond, an ester bond, a thioester bond, a urea bond, including any derivative or a combination thereof). In some embodiments, the linker of the invention comprises a click reaction product (e.g., a covalent linkage such as a cyclization reaction product, and/or a succinimide-thioether moiety formed via a click reaction). Additional linkers or spacers are well-known in the art.

Click reactions are well-known in the art and comprise inter alia Michael addition of maleimide and thiol (resulting in the formation of a succinimide-thioether); azide alkyne cycloaddition; Diels-Alder reaction (e.g., direct and/or inverse electron demand Diels Alder); dibenzyl cyclooctyne 1,3-nitrone (or azide) cycloaddition; alkene tetrazole photoclick reaction etc.

The term “heteroalkyl,” as used herein, refers to an alkyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups. Examples of heteroalkyl groups are polyglycols or polyalkoxylates, such as polyethyleneglycol.

In some embodiments, the derivatized porous MaSp-based fiber of the invention is as represented hereinbelow:

wherein a wavy bond represents a linker, wherein the linker is as described herein. In some embodiments, R is as described herein. In some embodiments, the linker is or comprises an optionally substituted C1-C6, or C1-C10 alkyl. In some embodiments, R comprises amino, halo, nitro, carbonyl, ester, or carboxy.

In some embodiments, the derivatized porous MaSp-based fiber of the invention is as represented hereinbelow:

wherein R is as described herein; n, m and o is are integers each independently being of between 0 and 20, wherein each R2 independently is or comprises H, or a substituent selected from halogen, —NO₂, —CN, —OH, —CONH₂, —CONR₂, —CNNR₂, —CSNR₂, —CONH—OH, —CONH—NH2, —NHCOR, —NHCSR, —NHCNR, —NC(═O)OR, —NC(═O)NR, —NC(═S)OR, —NC(═S)NR, —SO₂R, —SOR, —SR, —SO₂OR, —SO₂N(R)₂, —NHNR₂, —NNR, C₁-C₆ haloalkyl, optionally substituted C₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, hydroxy(C₁-C₆ alkyl), hydroxy(C₁-C₆ alkoxy), alkoxy(C₁-C₆ alkyl), alkoxy(C₁-C₆ alkoxy), C₁-C₆ alkyl-NR2, C₁-C₆ alkyl-SR, —CONH(C₁-C₆ alkyl), —CON(C1-C₆ alkyl)₂, —CO₂H, —CO₂R, —OCOR, —OCOR, —OC(═O)OR, —OC(═O)NR, —OC(═S)OR, —OC(═S)NR, or a combination thereof, and wherein X₁ is a heteroatom (e.g. O, S, N, NH) or is absent. In some embodiments, n, m and o are integers each independently being 0 or between 0 and 20, between 1 and 20, between 1 and 3, between 3 and 5, between 5 and 7, between 7 and 10, between 10 and 15, between 15 and 20, including any range between. In some embodiments, R is or comprises amino, carboxy,

or a combination thereof, wherein X₁ is as described herein, and R3 is or comprises H, optionally substituted C₁-C₁₀ alkyl, optionally substituted aryl, optionally substituted heteroaryl, or any combination thereof. In some embodiments, X₁ is or comprises N, or NH. In some embodiments, R2 is or comprises H, or an optionally substituted C₁-C₆ alkyl.

In some embodiments, the derivatized porous MaSp-based fiber of the invention is as represented hereinbelow:

wherein n, R2 and R3 are as described herein.

In some embodiments, the derivatized porous MaSp-based fiber of the invention is as represented hereinbelow:

wherein each R2 and n are as described hereinabove.

In some embodiments, the derivatized porous MaSp-based fiber of the invention is as represented hereinbelow:

wherein R and n are as described herein, and wherein each R₁ independently comprises any one of hydrogen, an alkyl (e.g. C₁-C₆ alkyl or C₁-C₁₀alkyl), hydroxy or C₁-C₁₀ alkoxy (e.g. methoxy, ethoxy, propoxy, pentoxy, etc.).

In some embodiments, the derivatized porous MaSp-based fiber of the invention is as represented hereinbelow:

wherein R1, n, R3 and R2 are as described herein.

In some embodiments, the functional moiety is covalently bound to any one of histidine, arginine, aspartate, glutamate or any combination thereof. One skilled in the art will appreciate, that the functional moiety can be covalently bound to a carboxylic side chain residue via an amino-, hydroxy- or mercapto-substituted linker.

In some embodiments, the derivatized porous MaSp-based fiber is as represented hereinbelow:

wherein X represents a sidechain heteroatom selected from O, S and N; and R is as described hereinabove. In some embodiments, X represents a heteroatom of any one of the amino acids of the MaSp-based fiber.

In some embodiments, the derivatized porous MaSp-based fiber is as represented hereinbelow:

wherein X represents a sidechain heteroatom of any one of the amino acids of the MaSp-based fiber;

represents an attachment point to the MaSp-based fiber, wavy bond represents a linker, and R is as described herein. In some embodiments, R comprises amino, halo, nitro, carbonyl, ester, or carboxy.

In some embodiments, the functional moiety is covalently bound to a hydroxy group of at least one tyrosine residue of the MaSp-based fiber.

In some embodiments, the derivatized porous MaSp-based fiber is as represented hereinbelow:

wherein X is as described herein.

In some embodiments, the functional moiety is covalently bound to at least one amino acid of the MaSp-based fiber via a silyl bond. In some embodiments, the amino acid is a nucleophilic amino acid (e.g. serine, cysteine, threonine and lysine). In some embodiments, the derivatized porous MaSp-based fiber is as represented hereinbelow:

wherein X represents a sidechain heteroatom (e.g. S, NH, or O) of any one of the amino acids of the MaSp-based fiber;

represents an attachment point to the MaSp-based fiber, wavy bond represents a linker, and R is as described herein, and each R₁ independently comprises any one of hydrogen, an alkyl, hydroxy or alkoxy.

In some embodiments, the derivatized porous MaSp-based fiber is as represented hereinbelow:

wherein X represents a sidechain heteroatom of any one of the amino acids of the MaSp-based fiber; and

represents an attachment point to the MaSp-based fiber. In some embodiments, X represents a sidechain heteroatom of cysteine and/or of tyrosine.

In some embodiments, the derivatized porous MaSp-based fiber is as represented hereinbelow:

wherein X represents a heteroatom selected from O, S and N; and R is as described hereinabove.

In some embodiments, the functional moiety is covalently bound to the MaSp-based fiber via at least one nucleophilic amino acid selected from serine, cysteine, threonine and lysine. In some embodiments, the functional moiety is covalently bound to a nucleophilic amino acid of the MaSp-based fiber via a bond selected from silyl, ester, carbamate, carbonyl, (O- or S)-thiocarbamate or a combination thereof.

In some embodiments, the functional moiety provides reactivity to the MaSp-based fiber. One skilled in the art will appreciate, that a functional moiety such as amine can react with an electrophile (e.g. haloalkyl or ester). Thus, by introducing a functional moiety to the MaSp-based fiber, the MaSp-based fiber can be subsequently reacted with any reagent (e.g. a small molecule or a polymer) having reactivity to the functional moiety. In some embodiments, the functional moiety induces or increases reactivity of the MaSp-based fiber. In some embodiments, the functional moiety induces or increases reactivity of the MaSp-based fiber towards any reagent capable of reacting therewith.

In some embodiments, the derivatized porous MaSp-based fiber of the invention comprises the functional moiety (e.g. amino or carboxy) covalently bound to a polymer. In some embodiments, the polymer is covalently attached to the functional moiety via a functional group having reactivity to the functional moiety (e.g. the functional moiety is or comprises carboxy, and the functional group of the polymer is amino, mercapto or hydroxy; or the functional moiety is or comprises amino, and the functional group of the polymer is halo, carbonyl, or carboxy). A skilled artisan will appreciate, that there are additional well-known reactive groups that can be utilized for the covalent attachment of the polymer to the functional moiety of the invention (e.g. via a click reaction). In some embodiments, the polymer is covalently attached to the functional moiety via any of —C(O)NH—, —C(O)O—, —C(O)—, —C(O)S—, —C(NH)NH—, —C(NH)O—, —C(NH)S—, —NC(O)—, —N(C)—, or a combination thereof.

In some embodiments, the functional moiety of the derivatized porous MaSp-based fiber is covalently bound to a polymer. In some embodiments, the polymer is positively and/or negatively charged. In some embodiments, the polymer is neutral.

In some embodiments, the polymer is selected from a cationic polymer (e.g. PEI, polylysine, polyarginine, chitosan) including any derivative and/or copolymer thereof), an anionic polymer (e.g. PAA) and/or a non-ionic polymer (e.g. PVA, PVC, silane, polyamide), etc. Other cationic polymers, anionic polymers, and/or non-ionic polymers are well-known in the art.

In some embodiments, the polymer is selected from the group consisting of polyglutaraldehyde (PGA), polyvinylaclohol (PVA), polyacrylic acid (PAA), polyacrylate, linear or branched polyethyleneimine (PEI), polyacrylamide (PAAm), polylysine, polyarginine, polyaniline, polyurethane, polyamide (e.g. nylon), polyvinyl chloride, polysilane, chitosan, N-halamine polymer, N-halamide polymer, polysilane-co-polyolefin, silane crosslinked polyolefin, and polyvinyl pyrrolidone (PVP) including any combination or copolymer thereof. In some embodiments, the polymer is a linear polymer. In some embodiments, the polymer is a branched polymer. In some embodiments, the polymer is a copolymer. In some embodiments, the polymer is a graft-copolymer.

In some embodiments, the polymer is covalently bound to a nucleophilic functional group or to an electrophilic functional group of the derivatized porous MaSp-based fiber. One skilled in the art will appreciate, that an aminated MaSp-based fiber can be reacted with a carboxy group of a polymer (e.g. PAA) or a carbonyl group of a polymer (e.g. PGA), so as to obtain the derivatized MaSp-based fiber covalently bound to a polymer. Furthermore, a carboxylated MaSp-based fiber can be reacted with amino group of a polymer (e.g. PEI) or with hydroxy group of a polymer (e.g. PVA). The inventors successfully synthesized conjugates of the derivatized MaSp-based fiber (e.g. comprising functional moieties bound to the tyrosine of the MaSp-based fiber via a diazo bond or via a silyl group, as described above) with various polymers such as PGA, PAA, PVA, PEI, PAAm or a combination thereof (such as PGA-co-PEI). Furthermore, the inventors successfully synthesized abovementioned conjugates using a MaSp-based protein having a mutant amino acid sequence (also used herein as “mutant MaSp-based protein”).

The inventors successfully synthesized a derivatized MaSp-based fiber modified with PAAm via in-situ polymerization on the aminated MaSp-based fiber represented by Formula 3:

In some embodiments, the derivatized MaSp-based fiber modified with PAAm is as represented below:

wherein each n is independently between 0 and 10, between 0 and 20, between 1 and 20, between 1 and 3, between 3 and 5, between 5 and 7, between 7 and 10, between 10 and 15, between 15 and 20, including any range between. In some embodiments, m is between 1 and 10000, between 1 and 10, between 10 and 100, between 100 and 1000, between 1000 and 10000, including any range between.

In some embodiments, the w/w ratio between the MaSp-based fiber and the polymer is 0.01:1 to 1:1, 0.02:1 to 1:1, 0.05:1 to 1:1, 0.09:1 to 1:1, 0.1:1 to 1:1, 0.5:1 to 1:1, or 0.9:1 to 1:1, including any range therebetween.

In some embodiments, the w/w ratio between the MaSp-based fiber and the polymer is 100:1 to 1:100, 95:1 to 1:100, 80:1 to 1:100, 60:1 to 1:100, 50:1 to 1:100, 30:1 to 1:100, 20:1 to 1:100, 10:1 to 1:100, 9:1 to 1:100, 5:1 to 1:100, 2:1 to 1:100, 100:1 to 1:80, 95:1 to 1:80, 80:1 to 1:80, 60:1 to 1:80, 50:1 to 1:80, 30:1 to 1:80, 20:1 to 1:80, 10:1 to 1:80, 9:1 to 1:80, 5:1 to 1:80, 2:1 to 1:80, 100:1 to 1:50, 95:1 to 1:50, 80:1 to 1:50, 60:1 to 1:50, 50:1 to 1:50, 30:1 to 1:50, 20:1 to 1:50, 10:1 to 1:50, 9:1 to 1:50, 5:1 to 1:50, 2:1 to 1:50, 100:1 to 1:10, 95:1 to 1:10, 80:1 to 1:10, 60:1 to 1:10, 50:1 to 1:10, 30:1 to 1:10, 20:1 to 1:10, 10:1 to 1:10, 9:1 to 1:10, 5:1 to 1:10, or 2:1 to 1:10, including any range therebetween.

In some embodiments, the functional group of the derivatized MaSp-based fiber is bound to a polymer comprising a plurality of reactive groups. In some embodiments, the reactive groups comprising a nucleophilic group (such as amino, hydroxy, thiol), an electrophilic group (such as carbonyl, carboxy, ester, succinimide ester, halo, nitro, and azide) or both. In some embodiments, a polyaldehyde-based polymer (e.g. poly-glutaraldehyde) bound MaSp-based fiber is as represented below:

wherein a dashed line represents an optional bond; and m and n are integers. In some embodiments, each n is independently between 0 and 10, between 0 and 20, between 1 and 20, between 1 and 3, between 3 and 5, between 5 and 7, between 7 and 10, between 10 and 15, between 15 and 20, including any range between. In some embodiments, m is between 1 and 10000, between 1 and 10, between 10 and 100, between 100 and 1000, between 1000 and 10000, including any range between.

In some embodiments, the derivatized MaSp-based fiber is bound to a polymer comprising a plurality of chelating agents. In some embodiments, the chelating agent comprises (i) metal chelating group capable of binding a metal or a salt thereof, (ii) a metal oxide chelating group, or both (i) and (ii).

In some embodiments, the metal chelating group is capable of complexing (via a coordinative bond) a metal or a salt thereof.

In some embodiments, the metal or the salt thereof comprises a transition metal. Non-limiting examples of transition metals include but are not limited to gold (Au), copper (Cu), palladium (Pd), zinc (Zn), aluminum (Al), tungsten (W), titanium (Ti), silicon (Si), zirconium (Zr), hafnium (Hf), hafnium (Hf), tin (Sn), gallium (Ga), molybdenum (Mo), nickel (Ni), vanadium (V), platinum (Pt), tantalum (Ta), germanium (Ge) and Niobium (Nb), or any combination thereof.

In some embodiments, the metal chelating group comprises a thiol, an amine, a phenol, a carboxy including any derivatives thereof. In some embodiments, the metal chelating group comprises a crown ether. In some embodiments, the metal chelating group is a cyclic molecule comprising a plurality of carboxy and/or hydroxy groups configured for complexing the metal or the salt thereof. In some embodiments, metal chelating group are well-known in the art comprising DOTA, NOTA, NODA, EDTA, HBED-CC including any salt, a derivative, or a combination thereof. In some embodiments, the metal chelating group comprises iminodiacetate (IDA), a salt or a derivative thereof.

In some embodiments, the polymer comprising a plurality of metal chelating groups is represented hereinbelow:

wherein CM represents a chelating agent; wherein k is from 10 to 10000, from 10 to 100, from 100 to 1000, from 100 to 10000, including any range between; m is from 0 to 10, from 0 to 3, from 3 to 5, from 5 to 10, including any range between; and each n represent an integer being independently between 0 and 10, between 0 and 20, between 1 and 20, between 1 and 3, between 3 and 5, between 5 and 7, between 7 and 10, between 10 and 15, between 15 and 20, including any range between.

In another aspect, the derivatized MaSp-based fiber comprises a functional moiety (e.g. a polymer) bound to a metal oxide chelating group, wherein the functional moiety is as described herein. In some embodiments, the metal oxide chelating group has an affinity to a metal oxide or to a particle comprising thereof. In some embodiments, the metal oxide chelating group is capable of complexing (e.g. via a coordinative bond) a metal oxide or a particle comprising thereof. In some embodiments, the metal oxide particle is as described herein.

In some embodiments, the term “complexing” refers to a stable (e.g. chemically stable) complexation of the metal and/or metal oxide, including any particle comprising thereof. In some embodiments, stable complexation refers to the ability of the derivatized MaSp-based fiber bound to the metal (also referred to herein as the “composite” or “stable composite”) retains at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of it's initial metal content upon exposure to a solvent (e.g. organic solvent, aqueous solvent, etc.), or storage under ambient conditions for a time period of for at least one month (m), at least 2 m, at least 6 m, at least 12 m, at least 2 years (y), at least 3y, at least 10y, including any range therebetween.

In some embodiments, the weight per weight (w/w) ratio of the functional moiety to the porous MaSp-based fiber within the derivatized fiber of the invention is between 0.01 and 30%, between 0.01 and 0.1%, between 0.1 and 0.5%, between 0.5 and 1%, between 1 and 5%, between 5 and 10%, between 10 and 20%, between 20 and 30%, including any range therebetween.

In some embodiments, loading of the functional moiety within derivatized porous MaSp-based fiber is between 0.01 μmol/g and 10 mmol/g, between 0.01 μmol/g and 0.1 μmol/g, between 0.1 μmol/g and 0.5 μmol/g, between 0.5 μmol/g and 1 μmol/g, between 1 μmol/g and 10 μmol/g, between 10 μmol/g and 30 μmol/g, between 30 μmol/g and 50 μmol/g, between 50 μmol/g and 100 μmol/g, between 100 μmol/g and 500 μmol/g, between 0.5 and 1 mmol/g, between 1 and 5 mmol/g, between 5 and 10 mmol/g, including any range therebetween.

In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% including any range therebetween, of the tyrosine residues within the derivatized MaSp-based fiber of the invention are substituted with the functional moiety, wherein the functional moiety is as described herein. In some embodiments, the substitution degree of the tyrosine residues within the derivatized MaSp-based fiber is between 1 and 99%, between 1 and 5%, between 5 and 10%, between 10 and 20%, between 20 and 30%, between 30 and 40%, between 40 and 50%, between 50 and 60%, between 60 and 70%, between 70 and 80%, between 80 and 90%, between 90 and 99%, including any range therebetween.

In some embodiments, the derivatized porous MaSp-based fiber of the invention comprises a functional moiety covalently bound to a side chain of at least one tyrosine so that the substitution degree of the tyrosine residues (or side chains) within the derivatized MaSp-based is between 1 and 90%, between 1 and 99%, between 10 and 99%, between 10 and 90%, between 10 and 80%, between 10 and 70%, between 10 and 60%, including any range or value therebetween.

In some embodiments, the substitution degree of the tyrosine residues (or side chains) within the derivatized MaSp-based fiber is at most 90%, at most 80%, at most 70%, at most 65%, at most 60%, including any range therebetween. The inventors successfully substituted up to about 60% of the tyrosine residues (or side chains) by various functional moieties, wherein some of the functional moieties are described in the examples section.

In some embodiments, the functional moiety is selectively bound to at least one tyrosine residue of the MaSp-based fiber. In some embodiments, selectively comprises at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% selectivity including any range therebetween.

In some embodiments, the functional moiety provides a positive and/or a negative charge to the MaSp-based fiber. In some embodiments, the functional moiety modifies a surface charge of the MaSp-based fiber. In some embodiments, the functional moiety modifies a property of the MaSp-based fiber, wherein the property is selected from wettability, water contact angle, dispersivity or solubility (e.g. in water and/or organic solvent).

In some embodiments, the derivatized (e.g. amino derivatized) porous MaSp-based fiber is characterized by a positive zeta-potential value being between 1 and 50 at pH of about 7. As exemplified herein, aminated MaSp-based fiber is characterized by a positive zeta-potential value of about 20 at pH of about 7, wherein the unmodified MaSp-based fiber has zeta-potential value of about −20 at pH of about 7.

In some embodiments, the derivatized (e.g. amino derivatized) porous MaSp-based fiber has greater zeta-potential value by at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 1000%, compared to zeta-potential value of the unmodified porous MaSp-based fiber.

In some embodiments, the derivatized (e.g. carboxy derivatized) porous MaSp-based fiber is characterized by a zeta-potential value being between −20 and −100 at pH of about 7. As exemplified herein, carboxylated MaSp-based fiber is characterized by zeta-potential value of about −40 at pH of about 7, wherein the unmodified MaSp-based fiber has zeta-potential value of about −20 at pH of about 7.

In some embodiments, the derivatized (e.g. carboxy derivatized) porous MaSp-based fiber has lower zeta-potential value by at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 1000%, compared to zeta-potential value of the unmodified porous MaSp-based fiber. One skilled in the art will appreciate, that the exact zeta-potential value will depend on pH and on the loading (i.e. w/w ratio) of the functional group within the porous MaSp-based fiber.

In some embodiments, the derivatized MaSp-based fiber retains the porosity of the pristine (e.g. non-derivatized) MaSp-based fiber. In some embodiments, the derivatized MaSp-based fiber is characterized by a BET surface area of at least 10 m²/g.

SEM images of the derivatized MaSp-based fibers are represented in FIGS. 3A and 3B. As demonstrated by FIGS. 3A-B, the outstanding porosity (e.g. defined by a BET surface area of at least 10 m²/g) of the derivatized MaSp-based fibers is sequence independent, since both MaSp-based protein and the mutant MaSp-based protein exhibit a highly porous structure. Furthermore, the porosity of the derivatized MaSp-based fibers is substantially retained, as compared to porosity of the non-modified MaSp-based fibers.

In some embodiments, the derivatized porous MaSp-based fiber of the invention comprises the functional moiety covalently bound to any one of a polymer, a linker and a chelating moiety or any combination thereof. In some embodiments, the functional moiety is covalently bound to a linker, wherein the linker is as described herein. In some embodiments, the functional moiety is covalently bound to a chelating moiety, wherein the chelating moiety is any one of a metal chelating group, a metal oxide chelating group or a combination thereof, wherein the metal chelating group and the metal oxide chelating group are as described herein.

In some embodiments, the derivatized porous MaSp-based fiber of the invention comprises wherein a dye or a pigment bound to the functional moiety, wherein the functional moiety is as described hereinabove. In some embodiments, the dye or the pigment is bound to the functional moiety via a covalent bond or a non-covalent bond. In some embodiments, the dye or the pigment is bound to the functional moiety via any one of hydrogen bond, Van-der Waals interaction, electrostatic interaction, p-p stacking or any combination thereof.

Non-limiting examples of dyes include but are not limited to: anionic dyes (e.g. Congo red, Alizarin Pure Blue B, Acid red 88, Trypan blue), cationic dyes (e.g. methine dyes, anthraquinone dyes, azo dyes, Coomassie, methylene blue), and neutral dyes (e.g. Neutral Orange RL, Neutral Red GRL, Neutral Gray 2BL), brilliant carmine 6B, lake red C, watching red, diazo yellow, hansa yellow, phthalocyanine blue, phthalocyanine green, alkali blue, and aniline black or any combination thereof. Other neutral or charged organic dyes are well-known in the art.

The inventors successfully implemented PEI and/or amino-modified derivatized fibers of the invention together with various dyes in hair coloring compositions, as described in the Examples section

In some embodiments, the composition of the invention comprises the derivatized porous MaSp-based fiber of the invention and optionally an additional component selected from an addition polymer, a dye and/or a pigment.

In some embodiments, the composition comprises 0.01% to 50%, 0.01% to 1%, 1% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 50% (w/w) of the MaSp-based fiber, including any range between; and an additional component.

In some embodiments, the composition comprises 0.001% to 95% (w/w), 0.005% to 95% (w/w), 0.009% to 95% (w/w), 0.01% to 95% (w/w), 0.05% to 95% (w/w), 0.09% to 95% (w/w), 0.1% to 95% (w/w), 0.5% to 95% (w/w), 0.9% to 95% (w/w), 1% to 95% (w/w), 5% to 95% (w/w), 10% to 95% (w/w), 15% to 95% (w/w), 20% to 95% (w/w), 30% to 95% (w/w), 50% to 95% (w/w), 0.01% to 80% (w/w), 0.05% to 80% (w/w), 0.09% to 80% (w/w), 0.1% to 80% (w/w), 0.5% to 80% (w/w), 0.9% to 80% (w/w), 1% to 80% (w/w), 5% to 80% (w/w), 10% to 80% (w/w), 15% to 80% (w/w), 20% to 80% (w/w), 30% to 80% (w/w), 50% to 80% (w/w), 0.001% to 50% (w/w), 0.005% to 50% (w/w), 0.009% to 50% (w/w), 0.01% to 95% (w/w), 0.01% to 50% (w/w), 0.05% to 50% (w/w), 0.09% to 50% (w/w), 0.1% to 50% (w/w), 0.5% to 50% (w/w), 0.9% to 50% (w/w), 1% to 50% (w/w), 5% to 50% (w/w), 10% to 50% (w/w), 15% to 50% (w/w), 20% to 50% (w/w), or 30% to 50% (w/w), of the derivatized porous MaSp-based fiber, including any range therebetween.

In some embodiments, the composition of the invention consists essentially of the derivatized porous MaSp-based fiber of the invention and optionally of the additional component, as described herein. In some embodiments, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, at least 99.9%, by weight of the composition, including any range between, consists of the derivatized porous MaSp-based fiber of the invention and optionally of the additional component, as described herein.

In some embodiments, the derivatized porous MaSp-based fiber consists essentially of any one of the derivatized porous MaSp-based fibers described herein. In some embodiments, the derivatized porous MaSp-based fiber of the invention is substantially devoid of an additional fiber (e.g. derivatized fiber), and/or of an additional polymer, and/or of an additional organic or inorganic material or particle.

In some embodiments, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, at least 99.9%, by weight of the derivatized porous MaSp-based fiber of the invention, including any range between, consists of any one of the derivatized porous MaSp-based fibers described herein.

In some embodiments, the derivatized porous MaSp-based fiber and/or the composition of the invention is stable. In some embodiments, the derivatized porous MaSp-based fiber of the invention is referred to as stable, if the derivatized fiber retains it's physical and/or chemical properties, and/or is chemically and/or physically stable upon dispersion in a solution, and/or prolonged storage under ambient storage conditions, and/or to a thermal exposure to a temperature up to 300° C., up to 200° C., up to 100° C., up to 80° C., up to 60° C., including any range between.

In some embodiments, the composition of the invention is referred to as stable, if the derivatized porous MaSp-based fiber of the invention is stably bound to the additional component (e.g. the composition is chemically stable upon dispersion in a solution, and/or prolonged storage under ambient storage conditions, and/or to a thermal exposure to a temperature up to 300° C., up to 200° C., up to 100° C., up to 80° C., up to 60° C., including any range between).

In some embodiments, the ambient conditions comprise exposure to any one of: an inert chemical such as a solvent (organic solvent and/or aqueous solvent, wherein the solvent is inert, i.e. is devoid of chemical reactivity with any of the components of the composition); a thermal exposure to a temperature up to 300° C., up to 200° C., up to 100° C., up to 80° C., up to 60° C., including any range between; exposure to UV/vis radiation (and/or electromagnetic radiation, IR radiation, microwave radiation, etc.); exposure to moisture and/or atmospheric gases, etc. In some embodiments, the ambient conditions comprise repetitive exposure to an inert chemical. In some embodiments, the ambient conditions comprise exposure to a temperature below the melting point and/or decomposition point of the any of the components of the composite (e.g. the MaSp-based fiber or of the derivatized MaSp-based fiber). One skilled in the art will appreciate that the exact definition of ambient storage conditions may include additional parameters or conditions well-known in the art.

In some embodiments, the composition and/or the derivatized porous MaSp-based fiber of the invention is referred to as stable, if it substantially maintains its structure, and its physical properties (e.g. mechanical stability, porosivity, tensile strength etc.) and chemical properties (wettability, zeta potential, hydrophobicity/hydrophilicity, reactivity), and/or wherein the additional component remains in contact with or bound to the derivatized MaSp-based fiber of the invention (e.g. substantially devoid of disintegration).

In some embodiments, the composition and/or the derivatized MaSp-based fiber of the invention is referred to as chemically stable if it substantially maintains it's chemical composition.

In some embodiments, the composition and/or the derivatized MaSp-based fiber of the invention is substantially chemically and/or physically stable for at least one month (m), at least 2 m, at least 6 m, at least 12 m, at least 2 years (y), at least 3y, at least 10y, including any range therebetween, wherein substantially is as described hereinbelow. In some embodiments, the composition and/or the derivatized MaSp-based fiber of the invention of the invention is substantially stable for a time period described herein, at ambient storage conditions.

Composite

In another aspect, there is provided a composite comprising the derivatized porous MaSp-based fiber of the invention bound to any one of a metal, a metal salt, or to a metal oxide particle or any combination thereof. In some embodiments, the metal oxide particle is bound to the derivatized porous MaSp-based fiber via a coordinative bond, via an electrostatic interaction or both.

In some embodiments, the composite of the invention comprises the derivatized porous MaSp-based fiber of the invention bound to a metal component selected from metal, a metal salt, or to a metal oxide or any combination thereof, wherein the metal component is in a form of a particulate matter or in a form of distinct atoms, and wherein the metal component is in an elemental state or in an oxidized state. In some embodiments, the metal component comprises a metal, and/or a metal salt, wherein the metal component comprises the first metal and/or the second metal of the invention.

In some embodiments, the composite of the invention comprises the derivatized porous MaSp-based fiber of the invention bound to a metal component as described herein, via one or more chelating agents of the invention.

In some embodiments, the metal component is coordinatively bound to the derivatized MaSp-based fiber via one or more chelating agents of the invention, and wherein the one or more chelating agents of the invention is covalently bound to the functional moiety of the invention (e.g. a polymer, as described herein). In some embodiments, the composite of the invention comprises the derivatized porous MaSp-based fiber of the invention bound to the metal component, wherein the derivatized porous MaSp-based fiber comprises a plurality of chelating agents covalently bound to the polymer, as described herein. In some embodiments, the chelating agent is as described hereinabove (e.g. comprising (i) metal chelating group capable of binding a metal or a salt thereof, (ii) a metal oxide chelating group, or both).

In some embodiments, the composite of the invention comprises a metal oxide particle bound to the derivatized porous MaSp-based fiber via a chelating agent. In some embodiments, the chelating agent is the metal oxide chelating group, as described hereinabove. In some embodiments, the metal oxide particle is complexed by the chelating agent. In some embodiments, the metal oxide particle is stably bound the derivatized porous MaSp-based fiber (e.g. the composite is chemically stable upon dispersion in a solution, and/or prolonged storage under ambient storage conditions, and/or to a thermal exposure to a temperature up to 300° C., up to 200° C., up to 100° C., up to 80° C., up to 60° C., including any range between).

In some embodiments, the ambient conditions comprise exposure to any one of: an inert chemical such as a solvent (organic solvent and/or aqueous solvent, wherein the solvent is inert, i.e. is devoid of chemical reactivity with any of the components of the composite); a thermal exposure to a temperature up to 300° C., up to 200° C., up to 100° C., up to 80° C., up to 60° C., including any range between; exposure to UV/vis radiation (and/or electromagnetic radiation, IR radiation, microwave radiation, etc.); exposure to moisture and/or atmospheric gases, etc. In some embodiments, the ambient conditions comprise repetitive exposure to an inert chemical. In some embodiments, the ambient conditions comprise exposure to a temperature below the melting point and/or decomposition point of the any of the components of the composite (e.g. the MaSp-based fiber or of the derivatized MaSp-based fiber). One skilled in the art will appreciate that the exact definition of ambient storage conditions may include additional parameters or conditions well-known in the art.

In some embodiments, the composite of the invention is referred to as stable, if it substantially maintains its structure, and its physical properties (e.g. mechanical stability, porosivity, tensile strength, electrical conductivity etc.) and chemical properties (wettability, zeta potential, hydrophobicity/hydrophilicity, reactivity), and/or wherein the metal component remains in contact with or bound to the derivatized MaSp-based fiber of the invention (e.g. substantially devoid of disintegration), wherein substantially is as described herein.

In some embodiments, the composite of the invention is referred to as chemically stable if the composite substantially maintains its chemical composition.

In some embodiments, the composite of the invention is substantially chemically and/or physically stable for at least one month (m), at least 2 m, at least 6 m, at least 12 m, at least 2 years (y), at least 3y, at least 10y, including any range therebetween, wherein substantially is as described hereinbelow. In some embodiments, the composite of the invention is substantially stable for a time period described herein, at ambient storage conditions.

In some embodiments, the composite of the invention comprises the derivatized MaSp-based fiber of the invention doped with the metal component. In some embodiments, the metal component is homogenously distributed within the composite (e.g. on or within the porous nanofibrils).

In some embodiments, the metal oxide is selected from the group consisting of titanium oxide, aluminum oxide, iron (II/III) oxide, zirconium oxide, zinc oxide, silicon oxide or any mixture thereof. In some embodiments, the metal oxide chelating group has an affinity and/or selectivity to titanium oxide. In some embodiments, the chelating agent has an affinity and/or selectivity to the metal oxide particle. In some embodiments, the chelating agent has an affinity and/or selectivity to a titanium oxide particle. In some embodiments, the metal oxide particle is as described hereinabove.

In some embodiments, the derivatized porous MaSp-based fiber comprises a functional moiety covalently bound to a metal oxide chelating group. In some embodiments, the metal oxide chelating group is covalently bound to the functional moiety via linker, wherein the linker is as described herein. In some embodiments, the metal oxide chelating group is covalently bound to the functional moiety via PGA linker. In some embodiments, each PGA chain is covalently bound to a plurality of metal oxide chelating groups.

In some embodiments, the functional moiety of the derivatized porous MaSp-based fiber covalently bound to a metal oxide chelating group is represented by Formula 4:

wherein a dashed line represents an optional bond, and each n is an integer being independently from 1 to 10000.

In some embodiments, the metal oxide chelating group comprises a carboxy and/or hydroxy group. In some embodiments, the metal oxide chelating group comprises a small molecule and/or a polymer. In some embodiments, the metal oxide chelating group is a mono-dentate, a bi-dentate, a tri-dentate, and a tetra-dentate ligand. In some embodiments, the metal oxide chelating group comprising a ligand having an affinity and/or selectivity to titania. In some embodiments, the metal oxide chelating group comprises a ligand comprising one or more carboxylic groups, and optionally one or more hydroxy groups. In some embodiments, the metal oxide chelating group comprises a ligand a cyclic multi-dentate ligand or a linear ligand.

In some embodiments, the metal oxide chelating group is a polymer comprising carboxy side chain groups and/or hydroxy side chain groups (such as PVA, polyacrylate, polyglycolate, etc., including any mixture or a copolymer thereof).

Non-limiting examples of metal oxide chelating groups include but are not limited to from salicylic acid, phosphonic acid, hydroxamic acid, malonic acid, pyrogallol, 5-hydroxy-1,4-naphtoquinone, quinone or any combination thereof. Other metal oxide chelating groups having affinity to titania are well-known in the art.

In some embodiments, the metal oxide chelating group comprises an oxidized tyrosine sidechain (e.g. dihydroxyphenyl or quinone). In some embodiments, the derivatized MaSp-based fiber comprises at least one oxidized tyrosine residue (e.g. in a form of dihydroxyphenyl or quinone). One skilled in the art will appreciate, that oxidized tyrosine can be obtained for example by reacting a MaSp based fiber with tyrosinase, thereby obtaining at least a portion of tyrosine residues being in an oxidized state (e.g. in a form of dihydroxyphenyl or quinone).

In some embodiments, the metal oxide chelating group is bound to a polymer, wherein the polymer is described herein. In some embodiments, the metal oxide chelating group is covalently bound to a polymer, wherein the polymer is as described herein. In some embodiments, the chelating moiety is covalently bound to the functional group of the derivatized MaSp-based fiber.

In some embodiments, the composite of the invention comprises the derivatized porous MaSp-based fiber comprising a plurality metal oxide chelating groups bound thereto, wherein at least a part (e.g. between 20 and 99%, between 20 and 30%, between 30 and 40%, between 40 and 60%, between 60 and 80%, between 80 and 90%, between 90 and 99%, including any range between) of the metal oxide chelating groups is bound to the metal oxide (e.g. metal oxide particle of the invention).

One skilled in the art will appreciate, that there are many options of covalent binding of the chelating moieties (e.g. metal oxide chelating groups) to a polymer. For example, an aminated chelating moiety can be bound to a carboxylated MaSp-based fiber. Alternatively, a carboxylated chelating moiety can be bound to an aminated MaSp-based fiber. Additionally, an aminated chelating moiety can be bound to a polymer comprising a carboxy group or a carbonyl group (e.g. PGA). Alternatively, a carboxylated chelating moiety can be bound to a polymer comprising a hydroxy group or an amino group (e.g. polylysine or PEI). The inventors successfully synthesized an aminated MaSp-based fiber bound to PGA, wherein PGA is further bound to a metal oxide chelating group (salicylic acid), as represented hereinabove.

In some embodiments, the composite of the invention comprises the derivatized porous MaSp-based fiber comprising a functional moiety covalently bound to a metal chelating group. In some embodiments, the metal chelating group is covalently bound to the functional moiety via linker, wherein the linker is as described herein. In some embodiments, the metal chelating group is covalently bound to the functional moiety via PGA linker. In some embodiments, each PGA chain is covalently bound to a plurality of metal chelating groups, as described above.

In some embodiments, the composite of the invention comprises the derivatized porous MaSp-based fiber comprising a plurality metal chelating groups bound thereto, wherein at least a part (e.g. between 20 and 99%, between 20 and 30%, between 30 and 40%, between 40 and 60%, between 60 and 80%, between 80 and 90%, between 90 and 99%, including any range between) of the metal chelating groups is bound to a metal, wherein the metal is as described herein. In some embodiments, at least a part of the metal chelating groups is bound to Pd or to a salt thereof (such as Pd(acetate)₂, PdCl₂). In some embodiments, at least a part of the metal chelating groups is bound to the metal at the elemental state (e.g. ground state, also referred to as 0 oxidation state). In some embodiments, at least a part of the metal chelating groups is bound to the metal at the oxidized state (e.g. +2). In some embodiments, the metal chelating groups is bound to at least partially reduced metal.

In some embodiments, the composition of the invention comprises the derivatized fiber of the invention doped with a metal (a first and/or a second metal, as described herein), wherein the metal is coordinatively bound to the polymer comprising a plurality of metal chelating groups. In some embodiments, the composition of the invention comprises Pd complexed by polyglutaraldehyde comprising a plurality of metal chelating groups (such as IDA), wherein the polyglutaraldehyde is covalently bound to the derivatized MaSp-based fiber of the invention.

In some embodiments, the polymer comprising a plurality of metal chelating groups complexing a metal or a metal cation is represented below:

wherein k is from 10 to 10000, M represents the transition metal (e.g. Pd or a salt thereof) and each m and n represent an integer being independently from 0 to 10.

In another aspect of the invention, the metal atom (or the first metal) complexed by the polymer of the invention is further bound to an additional metal atom, wherein the additional metal atom is the same or different. In some embodiments, the metal atom complexed by the polymer of the invention is further bound to a plurality of metal atoms, thereby forming an aggregate. In some embodiments, the first metal (e.g. Pd) complexed by the polymer of the invention is uniformly distributed on the outer surface of the derivatized fiber of the invention. In some embodiments, the first metal (e.g. Pd) forms a metal layer (or first metal layer) on top of the derivatized fiber of the invention. In some embodiments, the metal layer (e.g. first metal layer and/or second metal layer) is 1 to 10 atoms thick, including any range between. In some embodiments, the first metal (e.g. Pd) is in a colloidal form. In some embodiments, the first metal (e.g. Pd) is in a form of particles in a range between 1 and 500 nm.

In some embodiments, the first metal either in a form of a particulate or in a form of distinct atoms, forms an aggregation center appropriate for binding of a second metal on top thereof. In some embodiments, Pd (in the elemental state) complexed by the polymer of the invention as described herein, forms an aggregation center so as to allow deposition of the second metal (e.g. Cu). In some embodiments, the second metal is deposited via electroless plating. In some embodiments, the first metal is capable of facilitating electroless deposition of the second metal.

In some embodiments, the second metal (e.g. Cu) is in a colloidal form. In some embodiments, the second metal (e.g. Cu) is in a form of particles in a range between 1 and 500 nm, between 1 and 10 nm, between 10 and 50 nm, between 50 and 100 nm, between 100 and 200 nm, between 200 and 500 nm, including any range between.

In some embodiments, the second metal either in a form of a particulate matter or in a form of distinct atoms, wherein the distinct atoms are in an elemental state or in an oxidized state. In some embodiments, the second metal either in a form of a particulate matter or in a form of distinct atoms is homogenously distributed on top of the derivatized fiber of the invention. In some embodiments, the first metal and/or the second metal are in a form of a homogenous layer on top of the derivatized fiber of the invention. In some embodiments, the first metal and/or the second metal are in an amorphous state or in a crystalline state (e.g. forming a substantially crystalline particle). In some embodiments, at least a portion of the first metal and/or the second metal is in a crystalline state.

By “uniform” or “homogenous” it is meant to refer to size (or thickness) distribution that varies within a range of less than e.g., +60%, +50%, ±40%, +30%, +20%, or ±10%, including any value therebetween.

In some embodiments, the term “layer”, refers to a substantially uniform-thickness of a substantially homogeneous substance. In some embodiments, the shell comprises a single layer, or a plurality of layers.

In some embodiments, the derivatized MaSp-based fiber comprises a layer of the second metal (e.g. Cu⁰) deposited on top of or bound to the first metal (e.g. Pd), wherein the first metal is complexed by polyglutaraldehyde covalently bound to the MaSp-based fiber. In some embodiments, the first metal (e.g. Pd) is chelated or complexed by a metal chelating moiety (e.g. IDA). In some embodiments, the metal chelating moiety is covalently bound to polyglutaraldehyde as exemplified hereinabove. The inventors successfully doped the derivatized MaSp-based fiber with Cu⁰, wherein the derivatized MaSp-based fiber comprises PGA bound to IDA metal chelating group.

In some embodiments, the first metal and/or the second metal, as described herein are in a form of a form of a particulate matter or in a form of distinct atoms, and wherein each of the first metal and the second metal is independently in an elemental state or in an oxidized state.

In some embodiments, the metal layer (e.g. the first metal layer) in contact with or bound to the polymeric layer forms a plurality of aggregation sites for the second metal. In some embodiments, the first metal has a high affinity to the coating polymer of the polymeric layer. In some embodiments, the first metal is bound to the coating polymer.

In some embodiments, the second metal is bound to or aggregated on top of the first metal. In some embodiments, the second metal is in an elemental state. In some embodiments, the second metal is in an oxidized state (e.g. +1, or +2). In some embodiments, the second metal forms a layer on top of the first metal layer. In some embodiments, the second metal has an affinity to the first metal. In some embodiments, the second metal and the first metal are in a form of a layered structure, wherein each metal layer is separated. In some embodiments, the second metal and the first metal are mixed together within the metal layer. In some embodiments, the second metal and the first metal are mixed together, so as to form one metal layer on top of the polymeric layer. In some embodiments, the fiber is at least partially coated by any of the first metal and of the second metal or by a combination of the first and the second metal.

In some embodiments, the first metal and optionally the second metal are transition metals, as described herein. Transition metals are well-known in the art and are referred to metals comprising d-electrons.

In some embodiments, the first metal has a reduction potential suitable for a chemical reduction. In some embodiments, the first metal and the second metal are compatible with the electroless deposition method. In some embodiments, the first metal is capable of directly reducing the second metal.

In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% at least 99% of the chelating agents, including any range therebetween, are bound to the metal oxide particle.

In some embodiments, a w/w ratio of the derivatized porous MaSp-based fiber to the metal oxide particle is between 0.01:1 and 100:1, 0.01:1 to 10:1, 0.01:1 to 0.05:1, 0.05:1 to 0.1:1, 0.1:1 to 0.2:1, 0.2:1 to 0.3:1, 0.3:1 to 0.4:1, 0.4:1 to 0.5:1, 0.5:1 to 0.7:1, 0.7:1 to 0.9:1, 0.5:1 to 1:1, 0.9:1 to 1:1, 1:1 to 1.5:1, 1.5:1 to 2:1, 2:1 to 3:1, 3:1 to 5:1, 5:1 to 7:1, 7:1 to 10:1, 10:1 to 30:1, 30:1 to 50:1, 50:1 to 100:1, including any range therebetween.

In some embodiments, the metal oxide particle is characterized by a particle size between 25 and 5000 nm, between 25 and 50 nm, between 50 and 100 nm, between 100 and 150 nm, between 150 and 200 nm, between 200 and 300 nm, between 300 and 500 nm, between 500 and 1000 nm, between 1000 and 2000 nm, between 2000 and 3000 nm, between 3000 and 4000 nm, between 4000 and 5000 nm, including any range between.

In some embodiments, the composite is stable for at least 1 month (m), at least 2 m, at least 3 m, at least 4 m, at least 5 m, at least 6 m, at least 7 m, at least 8 m, at least 9 m, at least 10 m, at least 12 m, including any range therebetween.

In some embodiments, the metal oxide within the metal oxide particle is in an amorphous state. In some embodiments, the metal oxide within the metal oxide particle is in a crystalline state. In some embodiments, at least a portion of the metal oxide within the metal oxide particle is in an amorphous state.

In some embodiments, the composite is characterized by an increased dispersibility in an aqueous solution and/or organic solution, compared to a pristine (e.g. being devoid of MaSp-based fiber) metal oxide particle. A composite of the invention comprising titanium oxide particles having a particle size of above 300 nm exhibited a significantly improved dispersibility in an aqueous solution and/or organic solution, compared to pristine titania particles. An aqueous dispersion comprising titanium oxide particles bound to salicylate- or PVA-derivatized MaSp-based fiber (e.g. exemplary composites of the invention) demonstrated superior stability over a control dispersion comprising non-derivatized MaSp-based fibers. Furthermore, a composite comprising titanium oxide particles bound to salicylate-derivatized MaSp-based fibers demonstrated superior dispersibility (e.g. capable of forming a stable dispersion), wherein a w/w ratio of the titanium oxide particles to the derivatized MaSp-based fibers is about 1:1.

In some embodiments, an aqueous dispersion formed by the composite of the invention further comprises a surfactant. Exemplary surfactants, which have been implemented for the formation of stable dispersion include but are not limited to TRITON and sodium dodecyl sulfate (SDS).

In some embodiments, the composite is characterized by an increased absorption of a UV-radiation, compared to a control. In some embodiments, the control is a derivatized or a non-derivatized MaSp-based fiber devoid of metal oxide particles.

In some embodiments, the composite of the invention is characterized by electrical conductivity (also referred to herein as “conductivity”), wherein the composite is or comprises the derivatized MaSp-based bound to or doped by the metal (e.g. a metal in an elemental state, as described herein). In some embodiments, the metal induces or enhances electrical conductivity of the composite of the invention. In some embodiments, the derivatized MaSp-based bound to or doped by the metal (e.g. a metal in an elemental state, as described herein) is characterized by an improved electrical conductivity, compared to the derivatized MaSp-based fiber devoid of the metal. In some embodiments, a polymer enriched with the derivatized MaSp-based fiber (e.g. between 1 and 30%, between 1 and 10%, between 10 and 20%, between 20 and 30% enrichment by total weight of the composite, including nay range between), is characterized by an improved electrical conductivity, compared to the pristine polymer (e.g. being devoid of the derivatized MaSp-based fiber).

In some embodiments, the pristine MaSp-based fiber (e.g. non-derivatized fiber) and/or the derivatized MaSp-based fiber devoid of a metal is substantially non-conductive. In some embodiments, the pristine MaSp-based fiber (e.g. non-derivatized fiber) and/or the derivatized MaSp-based fiber devoid of a metal is characterized by a volume resistivity of at least 10¹³ ohm*cm, at least 10¹⁴ ohm*cm, at least 10¹⁵ ohm*cm, including any range between.

In some embodiments, the electrical conductivity of the composite is greater than the electrical conductivity of the pristine derivatized MaSp-based fiber (e.g. devoid of metal bound thereto) by at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, at least 1000000000% including any range therebetween.

In some embodiments, the electrical conductivity of the composite is by at least 10, at least 100, at least 1000, at least 10000, at least 100.000 times, at least 1,000,000 times, at least 10,000,000 times greater than the electrical conductivity of the pristine derivatized MaSp-based fiber, including any range therebetween.

In some embodiments, the composite of the invention is characterized by resistivity of between 10¹² and 10⁻⁵ ohm/m, between 10¹² and 10¹⁰ ohm/m, between 10¹⁰ and 10⁸ ohm/m, between 10⁸ and 10⁶ ohm/m, between 10⁶ and 10⁴ ohm/m, between 10⁴ and 10² ohm/m, between 10² and 1 ohm/m, between 1 and 10⁻⁵ ohm/m, between 1 and 10⁻² ohm/m, between 10⁻² and 10⁻³ ohm/m, between 10⁻³ and 10⁻⁵ ohm/m, including any range therebetween. In some embodiments, the resistivity is referred to an electrical resistivity normalized to the length of a sample.

In some embodiments, the derivatized MaSp-based fiber bound to or doped by the metal is characterized by anti-microbial activity, substantially reducing or preventing microbial load at the surface of the derivatized MaSp-based fiber, or at the surface of an additional polymer enriched with the derivatized MaSp-based fiber.

Method

In another aspect, there is provided a method of synthesizing the derivatized porous MaSp-based fiber of the invention, comprises reacting the porous MaSp-based fiber with a reagent represented by Formula I:

or by Formula II:

wherein R comprises the functional moiety of the invention; A is selected from aryl, heteroaryl, and alkyl substituted or non-substituted, including any combination thereof, each R₁ independently comprises any one of hydrogen, an alkyl, hydroxy or alkoxy, including any combination thereof, a wavy bond represents a linker or a bond; and wherein the reacting comprises conditions sufficient for covalent attachment of the functional moiety to the tyrosine of the porous MaSp-based fiber, thereby obtaining the derivatized porous MaSp-based fiber. In some embodiments, the linker is as described herein.

In some embodiments, at least one R₁ comprises a leaving group. In some embodiments, each R₁ independently comprises any one of hydrogen, or —O—C₁₋₁₀ alkyl. In some embodiments, A comprises a substituted or non-substituted aromatic ring. In some embodiments, A comprises a substituted or non-substituted phenyl.

In some embodiments, the linker comprises a substituted or non-substituted C₁₋₁₀ alkyl.

In some embodiments, the method of synthesizing the derivatized porous MaSp-based fiber of the invention, comprises providing a non-derivatized porous MaSp-based fiber and reacting thereof with a diazonium salt, as represented by Formula I, thereby covalently attaching the reagent via a diazo bond to the tyrosine of the MaSp-based fiber. In some embodiments, the diazonium salt is synthesized by reacting a nitrite salt with an amine, of Formula 5:

wherein R and A are as described herein. In some embodiments, the diazonium salt is synthesized via a reaction in an aqueous solution (e.g. an aqueous buffer). In some embodiments, the pH of the aqueous solution is of between 1 and 7, between 1 and 3, between 5 and 7, between 3 and 5, including any range between.

In some embodiments, the non-derivatized porous MaSp-based fiber is reacted with the reagent of Formula I in an aqueous solution (or aqueous suspension), or in a dispersion comprising a polar organic solvent. In some embodiments, the conditions sufficient for covalent attachment of the functional moiety to the tyrosine of the porous MaSp-based fiber comprise a pH of the aqueous solution is of between 3 and 10, between 5 and 7, between 3 and 5, between 7 and 10, including any range between. In some embodiments, the conditions sufficient for covalent attachment of the functional moiety to the tyrosine of the porous MaSp-based fiber comprise a temperature of less than 15° C., less than 10° C., less than 5° C., less than 3° C., including any range between.

In some embodiments, the method of synthesizing the derivatized porous MaSp-based fiber of the invention, comprises providing a non-derivatized porous MaSp-based fiber and reacting thereof with a reagent represented by Formula II, thereby covalently attaching the reagent via a silyl bond (C—Si bond) to the tyrosine of the MaSp-based fiber.

In some embodiments, the non-derivatized porous MaSp-based fiber is reacted with the reagent of Formula II in an aqueous solution (or aqueous suspension), or in a dispersion comprising a polar organic solvent. In some embodiments, the conditions sufficient for covalent attachment of the functional moiety to the tyrosine of the porous MaSp-based fiber comprise a pH of the aqueous solution is of between 6 and 12, between 5 and 7, between 6 and 10, between 7 and 10, between 7 and 9, between 9 and 12, including any range between. In some embodiments, the conditions sufficient for covalent attachment of the functional moiety to the tyrosine of the porous MaSp-based fiber comprise a temperature of less than 15° C., less than 10° C., less than 5° C., less than 3° C., including any range between.

In some embodiments, the method further comprises reacting the functional moiety (e.g. amine or carboxy) with a polymer having reactivity to the functional moiety, thereby covalently binding the polymer to the porous MaSp-based fiber; wherein the functional moiety comprises an amine or a carboxy group. In some embodiments, the functional moiety (e.g. amine) is reacted with a carboxy group of the polymer by addition of a coupling agent (such as HATU, HOBt, CDI, EDC, NHS, or a mixture thereof). In some embodiments, the functional moiety (e.g. carboxy) is reacted with an amine group of the polymer by addition of a coupling agent (such as HATU, HOBt, CDI, EDC, NHS, or a mixture thereof). Various coupling agents together with exact reaction conditions sufficient for amine-carboxy coupling are well-known in the art.

In some embodiments, the functional moiety (e.g. amine) is reacted with a carbonyl group (ketone or aldehyde) of the polymer, so as to form an imine. In some embodiments, the method further comprises reducing the imine to an amine, e.g. via sodium borohydride, or via hydrogen reduction together with an appropriate catalyst. Reaction conditions sufficient for imine bond formation and further imine-amine reduction are well-known in the art.

In some embodiments, the method further comprises reacting the polymer (e.g. PGA) with a chelating agent having reactivity to the polymer, thereby obtaining the chelating agent covalently bound to the polymer. In some embodiments, the method further comprises reacting the polymer (e.g. PGA) with a chelating agent, to obtain the chelating agent covalently bound to the polymer via an imine bond or via an amide bond, wherein the polymer comprises a carbonyl or carboxy group, respectively; and the chelating agent comprises an amine group.

Porous MaSp-Based Fiber

According to some embodiments, the present invention provides a composition comprising a derivatized porous MaSp-based fiber. In some embodiments, the derivatized MaSp-based fiber is present at a concentration of 0.1% to 90%, by total weight.

In some embodiments, the porous MaSp-based fiber comprises at least one MaSp-based fiber. In some embodiments, the at least one MaSp-based fiber is present at a concentration of 0.1% to 25%, 0.1% to 20%, 0.1% to 15%, 0.5% to 30%, 1% to 30%, 5% to 30%, or 10% to 30%, by total weight, including any range therebetween.

In some embodiments, the porous MaSp-based fiber is a MaSp-based polymer in the form of particles having a size in the range of 0.5 μm to 1.5 μm. In some embodiments, the MaSp-based fiber is an insoluble polymer. In some embodiments, the porous MaSp-based fiber has a DSC pattern exhibiting at least an endothermic peak in the range of from 200° C. to 280° C. In some embodiments, the porous MaSp-based fiber is characterized by an amide peak in the range of 1615 cm-1 to 1635 cm-1 as measured by FTIR analysis.

According to some embodiments, there is provided a composition comprising a MaSp-based polymer (e.g. synthetic MaSp-based polymer), wherein the MaSp-based polymer has at least one characterization selected from: a) being an insoluble polymer; b) being in the form of particles having a size in the range of 0.5 μm to 1.5 μm; c) a DSC pattern exhibiting at least an endothermic peak in the range of from 200° C. to 280° C.; and d) an amide peak in the range of 1615 cm-1 and 1638 cm-1 as measured by FTIR analysis.

In some embodiments, the MaSp-based fiber is characterized by a degradation temperature (T_(d)) between 280° C. and 350° C., between 290° C. and 350° C., between 300° C. and 350° C., between 310° C. and 350° C., between 320° C. and 350° C., between 280° C. and 330° C., between 290° C. and 330° C., between 300° C. and 330° C., between 310° C. and 330° C., or between 320° C. and 330° C., as determined by differential scanning calorimetry (DSC), including any range therebetween. Each possibility represents a separate embodiment of the invention.

In some embodiments, the MaSp-based fiber is characterized by a glass transition temperature (T_(g)) between 200° C. and 250° C., between 210° C. and 250° C., between 220° C. and 250° C., between 230° C. and 250° C., between 200° C. and 240° C., between 210° C. and 240° C., between 220° C. and 240° C., between 230° C. and 240° C., between 200° C. and 230° C., or between 210° C. and 230° C., as determined by DSC, including any range therebetween. Each possibility represents a separate embodiment of the invention.

In some embodiments, the MaSp-based fiber is characterized by a T_(g) between 260° C. and 320° C., between 270° C. and 320° C., between 280° C. and 320° C., between 290° C. and 320° C., between 260° C. and 310° C., between 270° C. and 310° C., between 280° C. and 310° C., or between 290° C. and 310° C., as determined by DSC, including any range therebetween. Each possibility represents a separate embodiment of the invention.

In some embodiments, the MaSp-based fiber is characterized by a DSC pattern exhibiting at least an endothermic peak between 280° C. and 350° C., between 290° C. and 350° C., between 300° C. and 350° C., between 310° C. and 350° C., between 280° C. and 330° C., between 290° C. and 330° C., between 300° C. and 330° C., between 310° C. and 330° C., or between 320° C. and 330° C., including any range therebetween. Each possibility represents a separate embodiment of the invention.

The term “degradation temperature (T_(d))” as used herein, refers to a temperature at which decomposition occurs. Thermal decomposition is a process of extensive chemical species change caused by heat.

As used herein, the term “glass transition temperature (T_(g))” refers to the temperature at which a material undergo a transition from a rubbery, viscous amorphous liquid (T>T_(g)), to a brittle, glassy amorphous solid (T<T_(g)). This liquid-to-glass transition (or glass transition for short) is a reversible transition. The glass transition temperature (T_(g)) is generally lower than the melting temperature (T_(m)), of the crystalline state of the material, if one exists.

According to some embodiments, there is provided the porous MaSp-based fiber comprising a synthetic MaSp-based polymer in the form of particles. In some embodiments, the particles have a size in the range of 0.5 μm to 1.5 μm, 0.7 μm to 1.5 μm, 0.8 μm to 1.5 μm, 0.9 μm to 1.5 μm, 0.5 μm to 1 μm, 0.7 μm to 1 μm, 0.8 μm to 1 μm, 0.9 μm to 1 μm, 0.5 μm to 1.3 μm, 0.5 μm to 1.2 μm, 0.7 μm to 1.3 μm, 0.7 μm to 1.2 μm, or 0.9 μm to 1.2 μm, including any range therebetween.

In some embodiments, the MaSp-based fiber comprises or consists of an insoluble MaSp-based polymer. In some embodiments, the insoluble MaSp-based polymer is in the form of particles. In some embodiments, the insoluble MaSp-based polymer is insoluble in organic solvents. In some embodiments, the insoluble MaSp-based polymer is insoluble in an aqueous solution. As used herein, the terms “MaSp-based polymer” and “MaSp-based fiber” are used herein interchangeably.

As used herein, the term “insoluble” refers to a material that, when exposed to an excess of solvent, does not dissolve, but may disperse to varying degrees. In some embodiments the term “insoluble” refers to a material that is less than 10%, less than 5%, less than 2%, or less than 1% soluble in a solvent. In some embodiments, “insoluble” refers to a material that can be partially dissolved in a solvent only at a concentration of less than 0.01% by weight. Solvents according to the present invention include organic solvents and aqueous solutions. In some embodiments, the solvent comprises an aqueous surfactant solution. In some embodiments, the solvent comprises urea aqueous solution.

In some embodiments, the MaSp-based fiber is characterized by a defined differential scanning calorimetry (DSC) pattern. In some embodiments, by “DSC pattern” it is meant to refer to the position of the peaks. In some embodiments, by “peak” it is meant to refer to exothermic peak. Herein throughout, “the position of the peaks” or “peak position” refers to the peaks along the temperature axis in a thermogram pattern, and, in some embodiments, may refers to the peak position at any peak intensity. One skilled in the art will appreciate that the data obtained in DSC measurements depend, in part, on the instrument used and the environmental conditions at the time measurements are carried out (e.g., humidity).

In some embodiments, the MaSp-based polymer is characterized by a DSC pattern exhibiting at least an endothermic peak in the range of from 200° C. to 280° C. In some embodiments, the disclosed composition is characterized by a DSC pattern exhibiting at least an endothermic peak in the range of from 200° C. to 270° C., 200° C. to 260° C., 200° C. to 250° C., 210° C. to 280° C., 212° C. to 280° C., 215° C. to 280° C., 216° C. to 280° C., 220° C. to 280° C., 210° C. to 250° C., 212° C. to 250° C., 215° C. to 250° C., 216° C. to 250° C., 220° C. to 250° C., 210° C. to 245° C., 210° C. to 242° C., or 215° C. to 245° C., including any range therebetween.

In some embodiments, the MaSp-based polymer is characterized by a DSC pattern exhibiting at least an endothermic peak with at least 5° C. to 100° C., at least 10° C. to 100° C., at least 15° C. to 100° C., at least 12° C. to 100° C., at least 25° C. to 100° C., at least 5° C. to 80° C., at least 10° C. to 80° C., at least 15° C. to 80° C., at least 12° C. to 80° C., at least 25° C. to 80° C., at least 5° C. to 50° C., at least 10° C. to 50° C., at least 15° C. to 50° C., at least 12° C. to 50° C., or at least 25° C. to 50° C., lower than the DSC pattern of an corresponding composition comprising a (MaSp)-based fiber.

In some embodiments, the MaSp-based polymer is devoid of DSC peaks in the range of about −100° C. to about 190° C. In some embodiments, the disclosed compound is devoid of DSC peaks in the range of about −100° C. to about 25° C. In some embodiments, the disclosed composition is characterized by at least a DSC pattern exhibiting devoid of an exothermic peak in the range of 40° C. to 70° C.

In some embodiments, the MaSp-based polymer is devoid of DSC peaks in the range of about −100° C. to about −50° C. In some embodiments, the disclosed compound is devoid of DSC peaks in the range of about −50° C. to about 0° C. In some embodiments, the disclosed compound is devoid of DSC peaks in the range of about −0° C. to about −25° C.

In some embodiments, the MaSp-based polymer is characterized by having an amide peak in the range of 1615 cm-1 to 1635 cm-1, as measured by FTIR analysis. In some embodiments, the disclosed composition is characterized by having an amide peak in the range of 1620 cm-1 to 1635 cm-1, 1620 cm-1 to 1630 cm-1, 1621 cm-1 to 1630 cm-1, or 1620 cm-1 to 1625 cm-1, including ay range therebetween, as measured by FTIR analysis.

In some embodiments, the MaSp-based polymer is devoid of a peak in the range of 1700 cm-1 to 1800 cm-1, as measured by FTIR analysis.

In one embodiment, the MaSp-based polymer of the invention assembles by self-assembly. By “self-assembly” it is meant that monomers, i.e., the synthetic spider silk protein of the invention, bind each other spontaneously, in an energetically favorable manner, under normal physiologic conditions, or at room temperature, to create the macromolecular structure having the properties described herein. Furthermore, the MaSp-based polymers of the invention are extremely resilient, and once assembled, may withstand extreme chemical assaults, such as solubilization in 10% surfactant solution and boiling for at least 1 hour.

“Tenacity” or “tensile strength” refers to the amount of weight a filament can bear before breaking. The maximum specific stress that is developed is usually in the filament, yarn or fabric by a tensile test to break the materials. According to specific embodiments, the MaSp-based polymer of the invention has tensile strength of about 100-3000 MPa (MPa=N/mm2), about 300-3000 MPa, about 500-2700 MPa, about 700-2500 MPa, about 900-2300 MPa, about 1100-2000 MPa, about 1200-1800 MPa, about 1300-1700 MPa or about 1400-1600 MPa. More specifically, about 1500 MPa.

“Toughness” refers to the energy needed to break the MaSp-based polymer. This is the area under the stress strain curve, sometimes referred to as “energy to break” or work to rupture. According to particular embodiments, the MaSp-based polymer of the invention a toughness of about 20-1000 MJ/m3, about 50-950 MJ/m3, about 100-900 MJ/m3, about 120-850 MJ/m3, about 150-800 MJ/m3, about 180-700 MJ/m3, about 180-750 MJ/m3, about 250-700 MJ/m3, about 280-600 MJ/m3, about 300-580 MJ/m3, about 310-560 MJ/m3, about 320-540 MJ/m3 or about 350-520 MJ/m3, most specifically about 350-520 MJ/m3.

“Elasticity” refers to the property of a body which tends to recover its original size and shape after deformation. Plasticity, deformation without recovery, is the opposite of elasticity. On a molecular configuration of the MaSp-based polymer, recoverable or elastic deformation is possible by stretching (reorientation) of inter-atomic and inter-molecular structural bonds. Conversely, breaking and re-forming of intermolecular bonds into new stabilized positions causes non-recoverable or plastic deformations.

“Extension” refers to an increase in length expressed as a percentage or fraction of the initial length.

By “fineness” is meant the mean diameter of a MaSp-based polymer or filament (e.g., a biofilament), which is usually expressed in microns (micrometers).

MaSp-Based Fibers

The terms “major ampullate spidroin protein” and “spidroin protein” are used interchangeably throughout the description and encompass all known major ampullate spidroin proteins, typically abbreviated “MaSp”, or “ADF” in the case of Araneus diadematus. These major ampullate spidroin proteins are generally of two types, 1 and 2. These terms furthermore include non-natural proteins, as disclosed herein, with a high degree of identity and/or similarity to at least the repetitive region of the known major ampullate spidroin proteins. Additional suitable spider silk proteins include MaSp2, MiSp, MiSp2, AcSp, FLYS, FLAS, and flagelliform.

As used herein, the term “repetitive region”, “repetitive sequence” or “repeat” refer to a recombinant protein sequence derived from repeat units which naturally occur multiple times in spider silk amino acid sequences (e.g., in the MaSp-1 protein). One skilled in the art will appreciate that the primary structure of the spider silk proteins is considered to consist mostly of a series of small variations of a unit repeat. The unit repeats in the naturally occurring proteins are often distinct from each other. That is, there is little or no exact duplication of the unit repeats along the length of the protein. In some embodiments, the synthetic spider silks of the invention are made wherein the primary structure of the protein comprises a number of exact repetitions of a single unit repeat. In additional embodiments, synthetic spider silks of the invention comprise a number of repetitions of one unit repeat together with a number of repetitions of a second unit repeat. Such a structure would be similar to a typical block copolymer. Unit repeats of several different sequences may also be combined to provide a synthetic spider silk protein having properties suited to a particular application. The term “direct repeat” as used herein is a repeat in tandem (head-to-tail arrangement) with a similar repeat. In another embodiment, the repeat used to form the synthetic spider silk of the invention is a direct repeat. In some embodiments, the repeat is not found in nature (i.e., is not a naturally occurring amino acid sequences).

An exemplary sequence comprising repetitive sequences is ADF-4: AAAAAAASGSGGYGPENQGPSGPVAYGPGGPVSSAAAAAAAGSGPGGYGPENQ GPSGPGGYGPGGSGSSAAAAAAAASGPGGYGPGSQGPSGPGGSGGYGPGSQGPS GPGASSAAAAAAAASGPGGYGPGSQGPSGPGAYGPGGPGSSAAASGPGGYGPGS QGPSGPGGSGGYGPGSQGPSGPGGPGASAAAAAAAAASGPGGYGPGSQGPSGPG AYGPGGPGSSAAASGPGGYGPGSQGPSGPGAYGPGGPGSSAAAAAAAGSGPGGY GPGNQGPSGPGGYGPGGPGSSAAAAAAASGPGGYGPGSQGPSGPGVYGPGGPGS SAAAAAAAGSGPGGYGPGNQGPSGPGGYGPGGSGSSAAAAAAAASGPGGYGPG SQGPSGPGGSGGYGPGSQGPSGPGASSAAAAAAAASGPGGYGPGSQGPSGPGAY GPGGPGSSAAASGPGGYGPGSQGPSGPGAYGPGGPGSSAAAAAAASGPGGYGPG SQGPSGPGGSRGYGPGSQGPGGPGASAAAAAAAAASGPGGYGPGSQGPSGPGYQ GPSGPGAYGPSPSASAS (SEQ ID NO: 1). In some embodiments, the synthetic repetitive sequence of the invention is based on (e.g., has a high percentage identity, as defined hereinbelow) one or more repetitive sequences derived from ADF-4 (SEQ ID NO: 1). As used herein, the term “based on” refers to a sequence having a high percentage of homology to a repetitive sequence.

In some embodiments, each repetitive sequence comprises up to 60 amino acids, up to 55 amino acids, up to 50 amino acids, up to 49 amino acids, up to 48 amino acids, up to 47 amino acids, up to 46 amino acids, up to 45 amino acids, up to 44 amino acids, up to 43 amino acids, up to 42 amino acids, up to 41 amino acids, up to 40 amino acids, up to 39 amino acids, up to 38 amino acids, up to 37 amino acids, up to 36 amino acids or up to 35 amino acids, wherein possibility represents a separate embodiment of the present invention. In some embodiments, each repetitive sequence comprises 5 to 60 amino acids, 10 to 55 amino acids, 15 to 50 amino acids, 20 to 45 amino acids, 25 to 40 amino acids, acids, 25 to 39 amino acids or 28 to 36 amino acids, wherein possibility represents a separate embodiment of the present invention. In some embodiments, each repetitive sequence comprises 30 to 40 amino acids, 31 to 39 amino acids, 32 to 38 amino acids, 33 to 37 amino acids, 34 to 36 amino acids, wherein each possibility represents a separate embodiment of the present invention. In an additional embodiment, each repetitive sequence comprises 35 amino acids.

In some embodiments, the repetitive region comprises, independently, an amino acid sequence as set forth in Formula 10 (X₁)_(z)X₂GPGGYGPX₃X₄X₅GPX₆GX₇GGX₈GPGGPGX₉X₁₀; wherein X₁ is, independently, at each instance A or G.

In some embodiments, at least 50% of (X₁)z is A, Z is an integer between 5 to 30; X₂ is S or G; X₃ is G or E; X₄ is G, S or N; X₅ is Q or Y; X₆ is G or S; X₇ is P or R; X₈ is Y or Q; X₉ is G or S; and X₁₀ is S or G.

In another embodiment, the repetitive region of a MaSP1 protein comprises the amino acid sequence as set forth in SEQ ID NO: 2 (SGPGGYGPGSQGPSGPGGYGPGGPGSS). In another embodiment, the repetitive region of a MaSP1 protein comprises the amino acid sequence as set forth in SEQ ID NO: 3 (AAAAAAAASGPGGYGPGSQGPSGPGGYGPGGPGSS).

In another embodiment, there is provided a homolog of the repetitive region of a MaSP1 protein sharing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with SEQ ID NO: 1.

In another embodiment, the homolog shares at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with SEQ ID NO: 2.

In another embodiment, the repetitive region of a MaSP1 protein has the amino acid sequence as set forth in SEQ ID NO: 1.

In another embodiment, the MaSP1 protein comprises a single N-terminal region selected from the group consisting of: SEQ ID NO: 4 (MSYYHHHHHHIDYDIPTTENLYFQGAMDPEFKGLRRRAQLV); SEQ ID NO: 5 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLVRPLSNLDNAP); SEQ ID NO: 6 (MSYYHIHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLVDPPGCRNSARAGS S); or any functional homolog, variant, derivative, or fragment thereof. In another embodiment, the homolog of the C-terminal region shares at least 70% homology with any one of SEQ ID NOs: 4-6.

In another embodiment, the MaSP1 protein further comprises a single C-terminal region selected from the group consisting of: SEQ ID NO: 7 (VAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCD ALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS); SEQ ID NO: 8 (GPSGPGAYGPSPSASASVAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALN SLVSQISASNPGLSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQA LS); or any functional homolog, variant, derivative, fragment or mutant thereof. In another embodiment, the homolog of the N-terminal region shares at least 70% homology with SEQ ID NO: 7-8.

In some embodiments, the MaSp-based fibers comprising a mixture of proteins, as disclosed under WO2017025964.

In some embodiments, the MaSp-based fiber comprises a mutant protein obtained by expressing a mutant nucleic acid sequence.

In some embodiments, the MaSP1 protein further comprises at least one tag sequence. Non-limiting examples of tags which may be used in the present invention include a His tag, a HA tag, a T7 tag, and the like. The skilled person is well aware of alternative suitable tags or other fusion partners.

MaSp-Based Fiber Comprising a Microbial Interacting Peptide

According to some embodiments, the MaSp-based fiber comprises a microbial interacting protein. In some embodiments, the microbial interacting protein is a virus binding receptor.

In some embodiments, the MaSp-based fiber comprises a peptide capable of interacting with a microbe (e.g., a virus). In some embodiments, the MaSp-based fiber comprises a peptide capable of binding a microbe (e.g., a virus).

In some embodiments, the microbial interacting protein is a virus binding receptor. In some embodiments, the virus binding receptor is a receptor used for viral entry. In some embodiments, the virus interacting protein is a host cell surface component recognized by the virus. In some embodiments, recognized by the virus is recognized as a gateway of entry into the cell.

In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is an alpha coronavirus. In some embodiments, the coronavirus is 229E or NL63. In some embodiments, the coronavirus is a beta coronavirus. In some embodiments the coronavirus is OC43, HKU1 or MERS-CoV. In some embodiments, the coronavirus is SARS-CoV-1. In some embodiments, the coronavirus is SARS-CoV-2.

In some embodiments, the virus binding receptor is angiotensin-converting enzyme 2 (ACE2). In some embodiments, the virus is a coronavirus and the virus binding receptor is angiotensin-converting enzyme 2 (ACE2).

In some embodiments, the ACE2 is a mutant ACE2. In some embodiments, the mutant ACE2 is a cleavage resistant ACE2 mutant.

Table 1 shows two non-limiting sequences that can be used as virus binding receptor.

Nucleotides Sequence Residues of ACE2 to introduce EEQAKTFLDKFNHEAEDLFYQSS-G- 22-44, 93 LGKGDFR (SEQ ID NO: 9) spacer of glycine, 351-357 AQMYPLQEIQN-G- 81-90, 129 EEQAKTFLDKFNHEAEDLFYQSS-G- spacer of glycine, LGKGDFR (SEQ ID NO: 10) 22-44, spacer of glycine, 351-357

In some embodiments, the MaSp-based fiber of the invention comprises an N-terminus, a repetitive region (e.g., 24 repeats), and a C-terminus. The repeats define the specific structure, and the C-terminus is important to the self-assembly. In some embodiments, the microbial interacting protein (e.g., virus binding receptor) can be inserted within the N-terminus region, with no or little effect on the structure (e.g., porosity) of the resulting protein.

A non-limiting example of the polynucleotide sequence encoding the virus binding receptor is

(SEQ ID NO: 11) GCCCAAATGTATCCACTACAAGAAATTCAGAATGGTGAGGAACAGGCCAA GACATTTTTGGACAAGTTTAACCACGAAGCCGAAGACCTGTTCTATCAAA GTTCAGGACTGGGGAAGGGCGACTTCAGG

Since the N-terminus of MaSp-based fiber of is a polylinker with multiple restriction sites, any restriction site that appears in the polylinker and does not appear in the insert may be used for the introduction of the polynucleotide sequence.

Table 2 provides a list of possible restriction enzymes for introducing a polynucleotide sequence encoding a virus binding receptor in the N-terminus region of the MaSp-based fiber.

Cut Cut Name Sequence Positions Name Sequence Positions EcoRV GATATC 39 Sall GTCGAC 101 HindIl GTYRAC 103 SgrDI CGTCGACG 101 Accl GTMKAC 102 Spel ACTAGT 114 Acyl GRCGYC 71 Styl CCWWGG 74 Asull TTCGAA 130 Tfil GAWTC 132 BamHl GGATCC 78 Xbal TCTAGA 135 Cfrl YGGCCR 121 Xholl RGATCY 78 EcoRI GAATTC 85 Agsl TTSAA 90 Narl GGCGCC 71 Haell RGCGCY 74 Ncol CCATGG 74 Sacl GAGCTC 111 Notl GCGGCCGC 121 Sdul GDGCHC 111

“Amino acid” as used herein, refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. “Amino acid analogs” refers to compounds that have the same fundamental chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

“Amino acid sequence” or “peptide sequence” is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins. The sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group Amino acid sequence is often called peptide, protein sequence if it represents the primary structure of a protein, however one must discern between the terms “Amino acid sequence” or “peptide sequence” and “protein”, since a protein is defined as an amino acid sequence folded into a specific three-dimensional configuration and that had typically undergone post-translational modifications, such as phosphorylation, acetylation, glycosylation, sulfhydryl bond formation, cleavage and the likes.

As used herein, “isolated” or “substantially purified”, in the context of synthetic spider silk amino-acid sequences or nucleic acid molecules encoding the same, as exemplified by the invention, means the amino-acid sequences or polynucleotides have been removed from their natural milieu or have been altered from their natural state. As such “isolated” does not necessarily reflect the extent to which the amino-acid sequences or nucleic acid molecules have been purified. However, it will be understood that such molecules that have been purified to some degree are “isolated”. If the molecules do not exist in a natural milieu, i.e. it does not exist in nature, the molecule is “isolated” regardless of where it is present. By way of example, amino-acid sequences or polynucleotides that do not naturally exist in humans are “isolated” even when they are present in humans.

The term “isolated” or “substantially purified”, when applied to an amino acid sequence or nucleic acid, denotes that the amino acid sequence or nucleic acid is essentially free of other cellular components with which they are associated in the natural state. It may be in a homogeneous state, or alternatively in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. An amino acid sequence or nucleic acid which is the predominant species present in a preparation is substantially purified.

In some embodiments, the repeats are of a homolog, variant, derivative of a repetitive region of a MaSp1 protein or fragment thereof. In some embodiments, the repeats are of a homolog, variant, derivative of a repetitive region of an ADF-4 protein or fragment thereof.

As used herein, the term “functional” as in “functional homolog, variant, derivative or fragment”, refers to an amino acid sequence which possesses biological function or activity that is identified through a defined functional assay. More specifically, the defined functional assay is the formation of self-assembling fibers in cells expressing the functional homolog, variant, derivative or fragment.

An amino acid sequence or a nucleic acid sequence is the to be a homolog of a corresponding amino acid sequence or a nucleic acid, when the homology is determined to be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99%.

Homology, as used herein, may be determined on the basis of percentage identity between two amino acid (peptide) or DNA sequences. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. The alignment of the two sequences is examined and the number of positions giving an exact amino acid (or nucleotide) correspondence between the two sequences determined, divided by the total length of the alignment multiplied by 100 to give a percentage identity figure. This percentage identity figure may be determined over the whole length of the sequences to be compared, which is particularly suitable for sequences of the same or very similar lengths and which are highly homologous, or over shorter defined lengths, which is more suitable for sequences of unequal length or which have a lower level of homology. Methods for comparing the identity of two or more sequences are well known in the art. Thus, for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1, for example the programs GAP and BESTFIT, may be used to determine the percentage identity between two amino acid sequences and the percentage identity between two polynucleotides sequences. BESTFIT uses the “local homology” algorithm of Smith and Waterman and finds the best single region of similarity between two sequences. BESTFIT is more suited to comparing two polypeptide or two polynucleotide sequences which are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer. In comparison, GAP aligns two sequences finding a “maximum similarity” according to the algorithm of Needleman and Wunsch. GAP is more suited to comparing sequences which are approximately the same length and an alignment is expected over the entire length. Preferably the parameters “Gap Weight” and “Length Weight” used in each program are 50 and 3 for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively. Preferably, percentage identities and similarities are determined when the two sequences being compared are optimally aligned.

The terms “identical”, “substantial identity”, “substantial homology” or percent “identity”, in the context of two or more amino acids or nucleic acids sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, or at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity over a specified region (e.g., amino acid sequence SEQ ID NO: 2 or 3), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then to be “substantially identical”. This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. The preferred algorithms can account for gaps and the like.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

It should be appreciated that the invention further encompasses amino acid sequence comprising 2-70 repeats of a variant of any one of SEQ ID NO: 1, 2, or 3. As used herein, the term “variant” or “substantially similar” comprises sequences of amino acids or nucleotides different from the specifically identified sequences, in which one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 25) amino acid residues or nucleotides are deleted, substituted or added. The variants may be allelic variants occurring naturally or variants of non-natural origin. The variant or substantially similar sequences refer to fragments of amino acid sequences or nucleic acids that may be characterized by the percentage of the identity of their amino acid or nucleotide sequences with the amino acid or nucleotide sequences described herein, as determined by common algorithms used in the state-of-the-art. The preferred fragments of amino acids or nucleic acids are those having a sequence of amino acids or nucleotides with at least around 40 or 45% of sequence identity, preferentially around 50% or 55% of sequence identity, more preferentially around 60% or 65% of sequence identity, more preferentially around 70% or 75% of sequence identity, more preferentially around 80% or 85% of sequence identity, yet more preferentially around 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity when compared to the sequence of reference.

In one embodiment, the MaSp-based polymer is a fiber.

In one embodiment, the MaSp-based polymer is composed of monomers. In one embodiment, a plurality of monomers are arranged in a nanofibril. In one embodiment, a plurality of nanofibrils are arranged in a fiber or make-up a fiber. In one embodiment, a monomer or a nanofibril within the MaSp-based polymer or a fiber has a diameter of 4 to 16 nm. In one embodiment, a monomer or a nanofibril within the MaSp-based polymer or a fiber has a diameter of 6 to 14 nm. In one embodiment, a monomer or a nanofibril within the MaSp-based polymer or a fiber has a diameter of 8 to 12 nm. In one embodiment, a fiber or the MaSp-based polymer has a diameter of 70 to 450 nm. In one embodiment, a fiber or the MaSp-based polymer of proteins has a diameter of 80 to 350 nm. In one embodiment, a fiber the MaSp-based polymer has a diameter of 80 to 300 nm. In one embodiment, a fiber or the MaSp-based polymer has a diameter of 150 to 250 nm. In one embodiment, a fiber or the MaSp-based polymer is arranged as a coil. In one embodiment, a single fiber or one the MaSp-based polymer is arranged as a coil. In one embodiment, a coil has a diameter of 5 to 800 micrometers. In one embodiment, a coil has a diameter of 5 to 500 micrometers. In one embodiment, a coil has a diameter of 5 to 30 micrometers. In one embodiment, a coil has a diameter of 5 to 20 micrometers. In one embodiment, a fiber or the MaSp-based polymer has a length of 5 to 800 micrometers. In one embodiment, a fiber or the MaSp-based polymer has a length of 30 to 300 micrometers.

In one embodiment, a fiber or the MaSp-based polymer is branched. In one embodiment, a fiber or the MaSp-based polymer comprises 1 to 10 branches. In one embodiment, a fiber or the MaSp-based polymer is free of carbohydrates. In one embodiment, a fiber or the MaSp-based polymer is non-glycosylated. In one embodiment, a fiber or the MaSp-based polymer is free of fat or fatty acids. In one embodiment, a fiber or the MaSp-based polymer is free of phosphorus. In one embodiment, a fiber or the MaSp-based polymer is free of an additional non-MaSp-based protein. In one embodiment, a fiber or the MaSp-based polymer is free of an additional polymer (e.g. a synthetic polymer, a non-MaSp-based peptide, non-MaSp-based protein). In one embodiment, a fiber or the MaSp-based polymer is substantially free of an additional polymer. In one embodiment, “free of” is “devoid of” or essentially “devoid of”.

In one embodiment, the aspect ratio of length to diameter of a fiber the MaSp-based polymer is at least 1:10. In one embodiment, the aspect ratio of length to diameter of a fiber or the MaSp-based polymer is at least 1:10 to 1:1500. In one embodiment, the aspect ratio of length to diameter of a fiber or the MaSp-based polymer is at least 1:50 to 1:1000. In one embodiment, the aspect ratio of length to diameter of a fiber or the MaSp-based polymer is at least 1:100 to 1:1200. In one embodiment, the aspect ratio of length to diameter of a fiber or the MaSp-based polymer is at least 1:100 to 1:1000. In one embodiment, the aspect ratio of length to diameter of a fiber or the MaSp-based polymer is at least 1:500 to 1:1000.

The terms derivatives and functional derivatives as used herein mean the amino acid sequence of the invention with any insertions, deletions, substitutions and modifications.

It should be appreciated that by the term “insertions”, as used herein it is meant any addition of amino acid residues to the sequence of the invention, of between 1 to 50 amino acid residues, specifically, between 20 to 1 amino acid residues, and more specifically, between 1 to 10 amino acid residues. Most specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 amino acid residues. Further, the amino acid sequence of the invention may be extended at the N-terminus and/or C-terminus thereof with various identical or different amino acid residues.

Amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In another embodiment, the repeat sequence of the invention has 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, or 7 or fewer amino acid substitutions to the sequence of any one of SEQ ID NO: 2 or 3. In one embodiment, the repeat sequence of the invention has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 amino acid substitutions to the sequence of any one of SEQ ID NO: 2 or 3.

With respect to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to an amino acid, nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues, and alleles of the invention.

For example, substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

Conservative nucleic acid substitutions are nucleic acid substitutions resulting in conservative amino acid substitutions as defined above.

Variants of the amino acid sequences of the invention may have at least 80% sequence similarity, at least 85% sequence similarity, 90% sequence similarity, or at least 95%, 96%, 97%, 98%, or 99% sequence similarity at the amino acid level, with a repeating unit denoted by any one of SEQ ID NO: 2 or 3.

The amino acid sequence of the invention may comprise 2-70 repeats of SEQ ID NO. 1 or SEQ ID NO. 3 or of any fragment thereof. A “fragment” constitutes a fraction of the amino acid or DNA sequence of a particular region. A fragment of the peptide sequence is at least one amino acid shorter than the particular region, and a fragment of a DNA sequence is at least one base-pair shorter than the particular region. The fragment may be truncated at the C-terminal or N-terminal sides, or both. An amino acid fragment may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 24, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33 or at least 34 amino acids of SEQ ID NO: 1 or 3.

Mutants of the amino acid sequences of the invention are characterized in the exchange of one (point mutant) or more, about up to 10, of its amino acids against one or more of another amino acid. They are the consequence of the corresponding mutations at the DNA level leading to different codons.

Still further, the invention concerns derivatives of the amino acid sequence of the invention. Derivatives of the amino acid sequences of the invention are, for example, where functional groups, such as amino, hydroxyl, mercapto or carboxyl groups, are derivatized, e.g. glycosylated, acylated, amidated or esterified, respectively. In glycosylated derivatives an oligosaccharide is usually linked to asparagine, serine, threonine and/or lysine. Acylated derivatives are especially acylated by a naturally occurring organic or inorganic acid, e.g. acetic acid, phosphoric acid or sulphuric acid, which usually takes place at the N-terminal amino group, or at hydroxy groups, especially of tyrosine or serine, respectively. Esters are those of naturally occurring alcohols, e.g. methanol or ethanol. Further derivatives are salts, especially pharmaceutically acceptable salts, for example metal salts, such as alkali metal and alkaline earth metal salts, e.g. sodium, potassium, magnesium, calcium or zinc salts, or ammonium salts formed with ammonia or a suitable organic amine, such as a lower alkylamine, e.g. triethylamine, hydroxy-lower alkylamine, e.g. 2-hydroxyethylamine, and the like.

In some embodiments, the silk protein of the invention is devoid of post translational modifications.

In some embodiments, the silk protein of the invention is biodegradable. This characteristic may be of importance, for example, in the field of medicine, whenever the silk proteins are intended for an in vivo use, in which biological degradation is desired. This characteristic may in particular find application in suture materials and wound closure and coverage systems.

According to some aspects, the MaSp-based fiber of the invention is manufactured using an expression vector comprising a suitable nucleic acid sequence, wherein the nucleic acid sequence is under expression control of an operably linked promoter and, optionally, regulatory sequences. Exemplary expression systems are known in the art, such as an expression system disclosed in PCT/IL2020/050752.

In some embodiments, the MaSp-based protein results in a self-assembled forming a defined structure. In some embodiments, the MaSp-based protein is in the form of a network. In some embodiments, the MaSp-based protein is in the form of a complex. In some embodiments, the MaSp-based protein induces a defined secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.

According to some aspects, the MaSp-based protein or the MaSp-based polymer which are used herein interchangeably, is in the form of a fiber. A “fiber” as used herein, is meant a fine cord of fibrous material composed of two or more filaments twisted together. By “filament” is meant a slender, elongated, threadlike object or structure of indefinite length, ranging from microscopic length to lengths of a mile or greater. Specifically, the synthetic spider silk filament is microscopic, and is proteinaceous. By “biofilament” is meant a filament created from a protein, including recombinantly produced spider silk protein. In some embodiments, the term “fiber” does not encompass unstructured aggregates or precipitates.

In some embodiments, the fiber of the proteins is characterized by size of at least one dimension thereof (e.g., diameter, length). For example, and without limitation, the diameter of the fiber is between 10 nm-1 μm, 20-100 nm, or 10-50 nm.

In some embodiments, the fiber is composed of nano-fibrils. In some embodiments, the nano-fibrils have a diameter of e.g., 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, about 25 nm, about 26 nm, about 27 nm, about 28 nm, about 29 nm, about 30 nm, about 31 nm, about 32 nm, about 33 nm, about 34 nm, about 35 nm, about 36 nm, about 37 nm, about 38 nm, about 40 nm, about 42 nm, about 44 nm, about 46 nm, about 48 nm, or about 50 nm, including any value or range therebetween. In one embodiment, the nano-fibrils have a diameter of 3-7 nm. In one embodiment, the nano-fibrils have a diameter of 4-6 nm.

In some embodiments, the length of the disclosed fiber is between 1-200 μm, 10-100 μm, 100 to 500 μm or 200-500 μm.

In some embodiments of any one of the embodiments described herein, the disclosed fiber (e.g. particle) is characterized by a porous structure. In some embodiments, the porous structure is characterized by a porosity of at least 30% (e.g., from 30 to 99%). In some embodiments, the porous structure is characterized by a porosity of at least 50% (e.g., from 50 to 99%). In some embodiments, the porous structure is characterized by a porosity of at least 60% (e.g., from 60 to 99%). In some embodiments, the porous structure is characterized by a porosity of at least 70% (e.g., from 70 to 99%). In some embodiments, the porous structure is characterized by a porosity of at least 80% (e.g., from 80 to 99%). In some embodiments, the porous structure is characterized by a porosity of at least 90% (e.g., from 90 to 99%). In some embodiments, the porous structure is characterized by a porosity of about 90%.

Herein, the term “porosity” refers to a percentage of the volume of a substance (e.g., a “sponge-like” material) which consists of voids. In another embodiment, porosity is measured according to voids or lumens within the surface area divided to the entire surface area (porous and non-porous).

In some embodiments, the porous structure of the disclosed fibers allows absorbing water efficiently on the fiber surface. That is, and without being bound by any particular theory, this surprising discovery can be explained in view of the disclosed fiber structure and its porosity which is in sharp distinction from native spider silk found in nature.

In some embodiments of any one of the embodiments described herein, the disclosed fiber is characterized by a mean diameter is nanosized.

In some embodiments, the disclosed fiber is characterized by a mean diameter is in a range of from 1 to 50 nm. In some such embodiments, the mean diameter is in a range of from 3 to 50 nm. In some such embodiments, the mean diameter is in a range of from 5 to 50 nm. In some such embodiments, the mean diameter is in a range of from 1 to 40 nm. In some such embodiments, the mean diameter is in a range of from 1 to 30 nm. In some such embodiments, the mean diameter is in a range of from 5 to 40 nm.

In some embodiments, the MaSp-based fiber comprises a plurality of pores. In some embodiments, the porous MaSp-based fiber comprises a plurality of fibrils (e.g. nano-fibrils), as exemplified hereinbelow (FIGS. 4A and 4B). In some embodiments, the MaSp-based fiber is a form of a particle, as described hereinbelow. In some embodiments, the MaSp-based fiber is as described hereinbelow. In some embodiments, the composition comprises a plurality of MaSp-based fibers. In some embodiments, the plurality of MaSp-based fibers comprises fibers having a different chemical composition and/or a different molecular weight (MW).

As further exemplified in the Examples section below, in some embodiments, a plurality of the disclosed fibers may be in the form of self-assembled structure or matrix. In some embodiments, this matrix can be rendered suitable for biomaterial applications.

In some embodiments, this matrix is suitable for cell growth, and for maintaining or promoting cellular activity, as further demonstrated hereinbelow.

In some embodiments, the term “self-assembled” refers to a resulted structure of a self-assembly process (e.g., spontaneous self-assembly process) based on a series of associative chemical reactions between at least two domains of the fiber(s), which occurs when the associating groups on one domain are in sufficient proximity and are oriented so as to allow constructive association with another domain. In other words, an associative interaction means an encounter that results in the attachment of the domains of a fiber or fibers to one another. In some embodiments, attached domains are not parallel to each other. Also contemplated are arrangements in which there are more than two domains of the self-assembled structure, each engaging a different plane.

It is noteworthy, that in some embodiments, the density of the self-assembled fiber (e.g., about 80% voids) is in the range of from 0.1 g/cm³ to 0.4 g/cm³ or from 0.2 g/cm³ to 0.3 g/cm³. In exemplary embodiments, the density of the self-assembled fiber is about 0.26 g/cm³.

Wettability study of surfaces at nano-scale spatial resolution and high temporal resolution is an emerging field from both theoretical and practical aspects. The disclosed fibers exhibited a high degree of surface's wettability exhibiting a remarkable ability to absorb fluids in comparison with their volume and weight.

In some embodiments, the polymer is hydrophobic. In some embodiments the polymer is UV cured.

In some embodiments, the disclosed composite is biostable. In some embodiments, the disclosed composite is biocleavable. In some embodiments, the disclosed composite is biodegradable.

In some embodiments, the term “biostable” describes a compound or a polymer that remains intact under physiological conditions (e.g., is not degraded in vivo, and hence is non-biodegradable or non-biocleavable).

In some embodiments, the term “biodegradable” describes a substance which can decompose under physiological and/or environmental condition(s) into breakdown products. Such physiological and/or environmental conditions include, for example, hydrolysis (decomposition via hydrolytic cleavage), enzymatic catalysis (enzymatic degradation), and mechanical interactions. This term typically refers to substances that decompose under these conditions such that 50 weight percent of the substance decompose within a time period shorter than one year.

In some embodiments, the term “biodegradable” as used in the context of embodiments of the invention, also encompasses the term “bioresorbable”, which describes a substance that decomposes under physiological conditions to break down products that undergo bioresorption into the host-organism, namely, become metabolites of the biochemical systems of the host-organism.

Anti-Microbial Composition

The present invention is based, in part, on the finding that a MaSp-based fiber and/or the derivatized MaSp-based fiber of the invention can be utilized for providing an increased anti-microbial effect by various means, including but not limited to, incorporation of a microbial-interacting protein and/or incorporation of at least one anti-microbial agent on the MaSp-based fiber having a nano-porous structure characterized by a large surface area.

The present invention is further based, in part, on the providing a MaSp-based fiber with increased wettability, thereby allowing increased incorporation of a droplet comprising a microbe on the fiber. Furthermore, the present invention is based, in part, on the providing a MaSp-based fiber incorporating therewith (e.g. via adsorption or via a covalent bond) an antimicrobial agent or a metal.

According to some embodiments, the present invention provides a composition comprising a MaSp-based fiber and/or the derivatized MaSp-based fiber of the invention comprising at least one anti-microbial agent. According to some embodiments, the anti-microbial agent and the MaSp-based fiber are bound via a hydrogen bond, Van der Waals bond, or both. According to some embodiments, the anti-microbial agent and the MaSp-based fiber are bound via hydrogen bonds, pi-pi electrostatic bonds, or both. According to some embodiments, the anti-microbial agent and the MaSp-based fiber and/or the derivatized MaSp-based fiber of the invention are bound via a covalent bond.

In some embodiments, the anti-microbial agent is selected from the group consisting of: a source of reactive oxygen species (ROS), a carboxylic acid, a quaternary amine, a disinfecting agent, a transition metal, an electrophilic reactive group, an oxidizing agent, an antimicrobial polymer or any combination thereof.

In some embodiments, ROS comprises oxygen-based reactive species. In some embodiments, ROS comprises peroxide, singlet oxygen, a peroxide, superoxide, hydroxyl radical, and alpha-oxygen or a combination thereof. ROS are responsible for the oxidation of the cellular envelope of microorganisms (e.g. bacteria and/or viruses).

In some embodiments, a source of ROS comprises a photosensitizer. In some embodiments, the photosensitizer is capable of in-situ generation of ROS upon activation by a light source. In some embodiments, the photosensitizer comprises Rose Bengal, malachite green, a cyanine dye, methylene blue, a porphyrin-based dye. Other photosensitizers are well-known in the art and are extensively utilized in Photodynamic Therapy of Cancer (PDT).

In some embodiments, a source of ROS comprises titania (TiO2). In some embodiments, titania is in a form of nano- or micro-particles. In some embodiments, titania particles have a diameter between 10 and 30 nm, between 30 and 100 nm, between 100 nm and 200 nm, between 200 and 500 nm, between 500 and 800 nm, between 800 nm and 1 um, between 1 and 5 um, between 5 and 10 um, between 10 and 100 um including any range therebetween.

TiO₂ particles generate electrons (e⁻) and positively charged holes (h⁺) in the conduction and valence bands of the semiconductor material, respectively, upon absorption of light in the UV-A region. In the presence of molecular oxygen, ROS (o₂ ⁻, HO₂ ⁻, H₂O₂ and, mainly, HO⁻) is generated subsequently.

In some embodiments, a source of ROS comprises a peroxide source. In some embodiments, the peroxide source is selected from the group consisting of: hydrogen peroxide, urea hydrogen peroxide, metal peroxide (such as sodium peroxide, calcium peroxide) and/or a derivative thereof, a percarbonate salt (such as sodium percarbonate, calcium percarbonate) and/or a derivative thereof, a periodate salt (such as sodium periodate) and/or a derivative thereof, a persulfate salt (such as sodium persulfate, ammonium persulfate) and/or a derivative thereof, a perborate salt (such as sodium perborate) and/or a derivative thereof, silver (II) oxide, perbenzoic acid and/or a derivative thereof (such as a chloro-perbenzoic acid, or a salt thereof), perchloric acid or a salt thereof, chlorine dioxide, benzoyl peroxide, a ketone peroxide, a peroxydicarbonate, a peroxyester, a dialkyl peroxide, a peroxyacetic acid (PAA), a hydroperoxide, a peroxyketal or any combination thereof.

In some embodiments, the carboxylic acid comprises a short-chain carboxylic acid. In some embodiments, the carboxylic acid comprises any of, propionic acid, acetic acid, butyric acid, an alpha-hydroxy carboxylic acid (e.g. lactic acid), citric acid, an amino acid or any combination thereof. In some embodiments, the oxidizing agent comprises a silver salt and hydrogen peroxide. In some embodiments, the oxidizing agent is peroxycarboxylic acid. In some embodiments, the oxidizing agent comprises hydrogen peroxide. In some embodiments, the oxidizing agent comprises hypochlorite, chlorite, chlorate, perchlorate, hexavalent chromium compounds (such as chromic and dichromic acids and chromium trioxide, pyridinium chlorochromate (PCC), dichromate), permanganate compounds (such as potassium permanganate), sodium perborate, nitrous oxide (N₂O), nitrogen dioxide, dinitrogen tetroxide (NO₂/N₂O₄), potassium nitrate (KNO₃), sodium bismuthate, cerium (IV) compounds (such as ceric ammonium nitrate and ceric sulfate) or any combination thereof.

In some embodiments, the electrophilic reactive group comprises any of aldehyde, ketone, oxime, acyl halide, an active ester (e.g. N-hydroxy succinimide), a chloroformate, an anhydride, a benzaldehyde (CHO), an epoxide, an isocyanate (such as a carbamate derivative of hexamethylene diisocyanate), a thiol (such as an ester derivative of thiopropionic acid), a benzaldehyde (such as an ester derivative of 4-formylbenzoic acid), an isothiocyanate, a maleimide, a carbonate, a sulfonyl chloride, a haloacetamide, an acyl azide, an imidoester, a carbodiimide, a vinyl sulfone, a thiol (SH), a C1-C10 thioalkyl, an ortho-pyridyl-disulfide, or any combination thereof.

In some embodiments, the antimicrobial polymer comprises a cationic polymer (chitosan, polylysine, and polyethyleneimine), N-halamine polymer, N-halamide polymer or any combination thereof. In some embodiments, the anti-microbial agent is an antimicrobial peptide (such as KLAKLAK, KALA, etc.).

In some embodiments, the anti-microbial agent comprises salicylic acid, chlorhexidine, benzalkonium chloride, ethanol, glutaraldehyde, formaldehyde, hydrogen peroxide, sodium hypochlorite.

In some embodiments, the anti-microbial agent is adsorbed to the MaSp-based fiber. In some embodiments, the anti-microbial agent is in contact with the MaSp-based fiber, so as to form a layer. In some embodiments, the layer is an outer layer. In some embodiments, the layer is in a form of a homogenous layer. In some embodiments, the MaSp-based fiber is coated by the anti-microbial agent. In some embodiments, the MaSp-based fiber is modified with the anti-microbial agent, wherein the anti-microbial agent is as described herein. In some embodiments, the fiber comprises the MaSp-based fiber and an additional polymer, wherein the additional polymer is as described herein.

In some embodiments, the composition comprises 0.01% to 50%, 0.01% to 1%, 1% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, and 20% to 50% (w/w) of the MaSp-based fiber, including any range between; and an additional polymer.

In another aspect of the invention, there is provided an antimicrobial composition. In some embodiments, the antimicrobial composition is configured to entrap (immobilize) the pathogen. In some embodiments, the antimicrobial composition is configured to prevent infestation (e.g. bacterial or viral infection). In some embodiments, the antimicrobial composition is configured to prevent from the pathogen to internalize into a cell (e.g. a human cell). In some embodiments, the antimicrobial composition is characterized by controlled-release properties with respect to one or more antimicrobial agents described herein (such as carboxylic acid).

In some embodiments, the antimicrobial composition comprising the MaSp-based fiber and/or the derivatized MaSp-based fiber of the invention; and the antimicrobial agent (such as glycolic acid and/or lactic acid) adsorbed thereto, is characterized by slow-release profile of the antimicrobial agent (glycolic acid and/or lactic acid). Such slow-release profile has been demonstrated by the inventors.

In some embodiments, the composition of the invention is for use in providing an anti-microbial effect to any one of a cream, foam, beads, gel, spray, film, non-woven mesh, a textile, and a substrate. In some embodiments, the composition of the invention is in a form of a coating.

In some embodiments, the composition of the invention comprises the derivatized MaSp-based fiber, wherein the functional moiety is or comprise a carboxylic acid. In some embodiments, the carboxylic acid has a polar or charged side chain, such as cysteic acid, thereby enhancing surface wettability of the fiber so as to form a superhydrophilic surface. In some embodiments, the surface of the MaSp based fiber and/or the derivatized MaSp-based fiber of the invention is at least partially modified with cysteic acid.

In some embodiments, the fiber surface is modified with cysteic acid covalently bound thereto, and further bound to an anti-microbial silver particle. In some embodiments, the silver particle has a diameter between 1 and 100 nm, between 10 and 10 nm, between 20 and 50 nm, between 50 and 100 nm including any range between. It is postulated, that a combination of a superhydrophilic surface (e.g. having a water-contact angle of less than 90°) with an anti-microbial silver particle is sufficient to induce inactivation of the captured microbes (e.g. viruses or bacteria).

In some embodiments, the composition of the invention comprises the derivatized MaSp-based fiber of the invention, wherein the functional moiety is or comprises an electrophilic reactive group (such as aldehyde, ketone, oxime, acyl halide, an active ester (e.g. N-hydroxy succinimide), a chloroformate, an anhydride, a benzaldehyde). In some embodiments, the electrophilic reactive group induces cross-linking of the pathogen (e.g. a virus and/or bacteria) entrapped or in contact with the fiber of the invention. In some embodiments, the electrophilic reactive group induces a covalent bond formation with the pathogen.

In some embodiments, the composition of the invention comprises the derivatized MaSp-based fiber of the invention, wherein the functional moiety is or comprises a monomer capable of polymerization (e.g. a lactone), and optionally a polymerization catalyst (e.g. Sn-based catalyst). In some embodiments, the composition of the invention induces polymerization of the monomer upon contacting with the pathogen. Such polymerization may comprise a formation of a polymeric chain (e.g. polyester such as PLA) in contact with the pathogen, so to entrap the pathogen on or within the fibrous composition of the invention. In some embodiments, the in-situ polymerized chain entraps and/or deactivates the pathogen.

In some embodiments, the composition of the invention comprises the derivatized MaSp-based fiber of the invention bound to the antimicrobial polymer (such as poly-glutaraldehyde). In some embodiments, the composition of the invention comprises the derivatized MaSp-based fiber in contact with or bound to an antimicrobial metal (such as copper, silver, zinc, nickel, cobalt, gold, or a combination thereof), or to an antimicrobial metal salt.

In some embodiments, the composition comprises 0.01% to 80% (w/w), 0.05% to 80% (w/w), 0.09% to 80% (w/w), 0.1% to 80% (w/w), 0.5% to 80% (w/w), 0.9% to 80% (w/w), 1% to 80% (w/w), 5% to 80% (w/w), 10% to 80% (w/w), 15% to 80% (w/w), 20% to 80% (w/w), 30% to 80% (w/w), 50% to 80% (w/w), 0.01% to 50% (w/w), 0.05% to 50% (w/w), 0.09% to 50% (w/w), 0.1% to 50% (w/w), 0.5% to 50% (w/w), 0.9% to 50% (w/w), 1% to 50% (w/w), 5% to 50% (w/w), 10% to 50% (w/w), 15% to 50% (w/w), 20% to 50% (w/w), or 30% to 50% (w/w), of the anti-microbial agent, including any range therebetween.

In some embodiments, the composition comprises 0.001% to 95% (w/w), 0.005% to 95% (w/w), 0.009% to 95% (w/w), 0.01% to 95% (w/w), 0.05% to 95% (w/w), 0.09% to 95% (w/w), 0.1% to 95% (w/w), 0.5% to 95% (w/w), 0.9% to 95% (w/w), 1% to 95% (w/w), 5% to 95% (w/w), 10% to 95% (w/w), 15% to 95% (w/w), 20% to 95% (w/w), 30% to 95% (w/w), 50% to 95% (w/w), 0.01% to 80% (w/w), 0.05% to 80% (w/w), 0.09% to 80% (w/w), 0.1% to 80% (w/w), 0.5% to 80% (w/w), 0.9% to 80% (w/w), 1% to 80% (w/w), 5% to 80% (w/w), 10% to 80% (w/w), 15% to 80% (w/w), 20% to 80% (w/w), 30% to 80% (w/w), 50% to 80% (w/w), 0.001% to 50% (w/w), 0.005% to 50% (w/w), 0.009% to 50% (w/w), 0.01% to 95% (w/w), 0.01% to 50% (w/w), 0.05% to 50% (w/w), 0.09% to 50% (w/w), 0.1% to 50% (w/w), 0.5% to 50% (w/w), 0.9% to 50% (w/w), 1% to 50% (w/w), 5% to 50% (w/w), 10% to 50% (w/w), 15% to 50% (w/w), 20% to 50% (w/w), or 30% to 50% (w/w), of the MaSp-based fiber, including any range therebetween.

In some embodiments, the ratio between the MaSp-based fiber and the anti-microbial agent is 0.01:1 to 1:1, 0.02:1 to 1:1, 0.05:1 to 1:1, 0.09:1 to 1:1, 0.1:1 to 1:1, 0.5:1 to 1:1, or 0.9:1 to 1:1, including any range therebetween.

In some embodiments, the ratio between the MaSp-based fiber and the anti-microbial agent is 100:1 to 1:100, 95:1 to 1:100, 80:1 to 1:100, 60:1 to 1:100, 50:1 to 1:100, 30:1 to 1:100, 20:1 to 1:100, 10:1 to 1:100, 9:1 to 1:100, 5:1 to 1:100, 2:1 to 1:100, 100:1 to 1:80, 95:1 to 1:80, 80:1 to 1:80, 60:1 to 1:80, 50:1 to 1:80, 30:1 to 1:80, 20:1 to 1:80, 10:1 to 1:80, 9:1 to 1:80, 5:1 to 1:80, 2:1 to 1:80, 100:1 to 1:50, 95:1 to 1:50, 80:1 to 1:50, 60:1 to 1:50, 50:1 to 1:50, 30:1 to 1:50, 20:1 to 1:50, 10:1 to 1:50, 9:1 to 1:50, 5:1 to 1:50, 2:1 to 1:50, 100:1 to 1:10, 95:1 to 1:10, 80:1 to 1:10, 60:1 to 1:10, 50:1 to 1:10, 30:1 to 1:10, 20:1 to 1:10, 10:1 to 1:10, 9:1 to 1:10, 5:1 to 1:10, or 2:1 to 1:10, including any range therebetween.

In another aspect of the invention, there is provided a composition or a article comprising the MaSp-based fiber in contact with or bound (e.g. non-covalently bound) to a metal. In some embodiments, the composition comprises a metal layered fiber, wherein the fiber comprises the MaSp-based fiber and optionally an additional polymer, as described herein. In some embodiments, the fiber is in contact or coated with a polymeric layer, and wherein the polymeric layer is in contact or coated with a metal, wherein the metal is as described herein. In some embodiments, the fiber is at least partially coated with the polymeric layer.

In some embodiments, the composition or the article comprises the additional polymer enriched with the MaSp-based fiber. In some embodiments, enrichment is between 1 and 50%, between 5-10%, between 10 and 15%, between 15 and 20% including any range therebetween. In some embodiments, the additional polymer enriched with the MaSp-based fiber is in a form a fiber or a thread. In some embodiments, the additional polymer enriched with the MaSp-based fiber is in a form a layer. In some embodiments, the additional polymer enriched with the MaSp-based fiber is in a form a fibrous mate.

In some embodiments, the composition or the article is a multi-layer composition, comprising the fiber coated with the polymeric layer, and wherein the polymeric layer is bound to an outer layer comprising a metal. In some embodiments, the polymeric layer is homogeneous. In some embodiments, the polymeric layer is in a form of a coating. In some embodiments, the polymeric layer partially coats the fiber.

In some embodiments, the polymer (also referred to herein as a coating polymer) is bound to or entrapped within the fibrils or pores of the MaSp-based fiber. In some embodiments, the coating polymer is electrostatically bound to the MaSp-based fiber. In some embodiments, the coating polymer is adhered to the MaSp-based fiber and/or to the fiber. In some embodiments, the coating polymer is adhered to the additional polymer comprising the fiber.

In some embodiments, the additional polymer is selected form the group consisting of thermoplastic polymer, a thermoset an epoxy, a polyester a polyamide, a polyol, a polyurethane, polyethylene, Nylon, a polyacrylate, a polycarbonate, polyaldehyde, polycarboxylic acid, polyamine, polyimine, polylactic acid (PLA) or a copolymer thereof a silicon, a liquid crystal polymer, a maleic anhydride grafted polypropylene, polycaprolactone (PCL), rubber, cellulose, or any combination thereof.

In some embodiments, there is provided an article comprising the composition described hereinabove. In some embodiments, the article is an anti-microbial article. In some embodiments, the article is a conductive device. In some embodiments, the article is a conductive substrate. In some embodiments, the article is in a form of a coated glass substrate.

In some embodiments, the article is a cosmetic article, comprising the derivatized MaSp based fibers of the invention (e.g. aminated, or PEI modified), having a positive zeta potential. In some embodiments, the article is a cosmetic article, comprising the composite of the invention (e.g. metal oxide particles complexed by the derivatized MaSp based fibers of the invention). In some embodiments, the article is an electronic device. In some embodiments, the article is an anti-bacterial coating. In some embodiments, the article is a woven or a non-woven mate. In some embodiments, the article is a fiber or thread enriched with the metal doped fiber of the invention.

In some embodiments, there is provided a kit comprising the composition or the composite of the invention. In some embodiments, the kit further comprises an additional material, e.g. a polymer, a glass, ceramics, a particle (e.g. a carbon particle) or a metal substrate.

The thickness of the polymeric layer is between 1 nm and 1 um, between 1 and 50 nm, between 1 and 10 nm, between 10 and 50 nm, between 50 and 100 nm, between 100 and 300 nm, between 300 and 500 nm, between 500 and 1000 nm including any range between. In some embodiments, the polymeric layer comprises the coating polymer.

In some embodiments, the polymeric layer comprises one or more polymers. In some embodiments, the coating polymer is compatible with the MaSp-based fiber (e.g. has a sufficient adhesion strength to the MaSp-based fiber). In some embodiments, the coating polymer is compatible with the additional polymer within the fiber. In some embodiments, the coating polymer is compatible with the metal. In some embodiments, the coating polymer is compatible with the metal and with the fiber of the invention.

In some embodiments, the coating polymer comprises polar atoms or polar groups. In some embodiments, the polar atoms or polar groups are so as to provide a sufficient affinity (i.e. adhesion strength) to the metal (such as Pd). In some embodiments, the coating polymer comprises atoms with electronegativity less than the electronegativity of carbon. In some embodiments, the coating polymer comprises any of N, O, S or a combination thereof. In some embodiments, the coating polymer comprises any of amino, hydroxy, carbonyl, carboxy, ester, ether, amide or a combination thereof.

In some embodiments, the coating polymer comprises any of polyethyleneimine (PEI), poly-lysine, polyarginine, polyester (e.g., PLA, PCL), polyamide (nylon), polyvinyl alcohol, polyether (PEG) or a combination thereof. In some embodiments, the coating polymer comprises PEI. In some embodiments, the coating polymer is bound to an additional polymeric layer.

In some embodiments, the polymeric layer is in contact with or bound to the metal. In some embodiments, the metal is uniformly distributed on the outer surface of the polymeric layer. In some embodiments, the metal forms a layer on top of the polymeric layer. In some embodiments, the metal layer is 1 to 10 atoms thick, including any range between. In some embodiments, the metal is in a colloid form. In some embodiments, the metal is in a form of particles in a range between 1 and 500 nm.

In some embodiments, the polymeric layer is in contact with or bound to a first metal layer comprising a first metal. In some embodiments, the polymeric layer is in contact with or bound to a first metal layer and to a second metal layer, wherein the second metal layer comprises a second metal. In some embodiments, the first metal and the second metal are the same. In some embodiments, the first metal and the second metal are different.

In some embodiments, the metal layer (e.g. the first metal layer) in contact with or bound to the polymeric layer forms a plurality of aggregation sites for the second metal. In some embodiments, the first metal has a high affinity to the coating polymer of the polymeric layer. In some embodiments, the first metal facilitates deposition of the second metal (e.g. by electroless plating). In some embodiments, the first metal is capable of reducing a salt of the second metal, thereby forming the second metal layer on top or in contact with the first metal. In some embodiments, the first metal is bound to the coating polymer.

In some embodiments, the second metal is bound to or aggregated on top of the first metal. In some embodiments, the second metal forms a layer on top of the first metal layer. In some embodiments, the second metal has an affinity to the first metal. In some embodiments, the second metal and the first metal are in a form of a layered structure, wherein each metal layer is separated. In some embodiments, the second metal and the first metal are mixed together within the metal layer. In some embodiments, the second metal and the first metal are mixed together, so as to form one metal layer on top of the polymeric layer. In some embodiments, the fiber is at least partially coated by any of the first metal and of the second metal or by a combination of the first and the second metal.

In some embodiments, the metal layer comprises a chemically reducible metal as the first metal, wherein chemically reducible refers to the reduction of the first metal salt by chemical reduction process. In some embodiments, the metal layer comprises an antimicrobial metal as the second metal.

In some embodiments, the metal layer is devoid of the second metal. In some embodiments, the metal layer comprises at least 90%, at least 95%, at least 99% of the first metal, wherein the first metal is an antimicrobial metal. In some embodiments, the first metal and optionally the second metal are transition metals. Transition metals are well-known in the art and are referred to metals comprising d-electrons.

In some embodiments, the antimicrobial metal comprises copper, silver, zinc or a combination thereof. In some embodiments, the first metal or a salt thereof has a reduction potential suitable for a chemical reduction. In some embodiments, the first metal and the second metal are compatible with the electroless deposition method.

In some embodiments, the composition of the invention comprises (i) the fiber comprising the MaSp-based and optionally the additional polymer (e.g. Nylon or PCL); wherein the fiber is coated with (ii) the polymeric layer comprising the coating polymer (e.g. PEI); and wherein the polymeric layer is in contact with or adhered to (iii) the metal layer comprising the first metal (e.g. Pd, Cu, Ag, Zn, and optionally a salt thereof) and further comprising the second metal, such as the antibacterial metal (e.g. Cu or Ag).

In some embodiments, the multi-layered fibrous composition of the invention is exemplified by FIG. 1 .

In some embodiments, the MaSp-based fiber or a composition comprising the MaSp-based fiber and the additional polymer (e.g. Nylon) facilitates metal binding thereto. In some embodiments, the MaSp-based fiber or a composition comprising the MaSp-based fiber and the additional polymer (e.g. Nylon) is characterized by an enhanced metal binding affinity, as compared to a polymer devoid of the MaSp-based fiber, as exemplified by FIG. 2 . In some embodiments, the fiber or the metal-coated fiber of the invention is characterized by an anti-microbial effect. In some embodiments, the fiber or the metal-coated fiber of the invention is characterized by an enhanced anti-microbial effect, as compared to other coated or non-coated polymers. In some embodiments, the anti-microbial effect is as exemplified by FIG. 4 .

In some embodiments, the composition of the invention is in a form of a coating on top of a substrate. In some embodiments, the composition of the invention forms a mixture with the substrate. In some embodiments, the composition of the invention is in a form of an antimicrobial additive (e.g. a mix or a coating). In some embodiments, the composition (e.g. the fiber) is characterized by a wettability. In some embodiments, the wettability is suitable for allowing contact of a droplet (e.g., comprising a viral particle) for a time sufficient to inactivate a microbe within the droplet. In some embodiments, the wettability is characterized by a contact angle below 100°, below 90°, below 80°, below 70°, below 60°, including any value between.

In some embodiments, the wettability is achieved by incorporating superhydrophobic particles or polar molecules (such as cysteic acid) on top of the MaSp-based fiber or within the composition. In some embodiments, the superhydrophobic particle comprises cysteic acid.

In some embodiments, the ratio between the substrate and the anti-microbial composition 1000:1 to 1:100, 1000:1 to 1:50, 1000:1 to 1:10, 1000:1 to 1:1, 900:1 to 1:100, 900:1 to 1:10, 900:1 to 1:1, 500:1 to 1:100, 500:1 to 1:10, 500:1 to 1:1, 100:1 to 1:100, 95:1 to 1:100, 80:1 to 1:100, 60:1 to 1:100, 50:1 to 1:100, 30:1 to 1:100, 20:1 to 1:100, 10:1 to 1:100, 9:1 to 1:100, 5:1 to 1:100, 2:1 to 1:100, 100:1 to 1:80, 95:1 to 1:80, 80:1 to 1:80, 60:1 to 1:80, 50:1 to 1:80, 30:1 to 1:80, 20:1 to 1:80, 10:1 to 1:80, 9:1 to 1:80, 5:1 to 1:80, 2:1 to 1:80, 100:1 to 1:50, 95:1 to 1:50, 80:1 to 1:50, 60:1 to 1:50, 50:1 to 1:50, 30:1 to 1:50, 20:1 to 1:50, 10:1 to 1:50, 9:1 to 1:50, 5:1 to 1:50, 2:1 to 1:50, 100:1 to 1:10, 95:1 to 1:10, 80:1 to 1:10, 60:1 to 1:10, 50:1 to 1:10, 30:1 to 1:10, 20:1 to 1:10, 10:1 to 1:10, 9:1 to 1:10, 5:1 to 1:10, or 2:1 to 1:10, including any range therebetween.

In some embodiments, the substrate comprises any one of a textile substrate, a fabric, a polymeric substrate, a glass substrate, and a metal substrate.

In another aspect of the invention, there is provided a composition comprising the fiber of the invention covalently bound to the anti-microbial agent, wherein the anti-microbial agent is as described herein.

In some embodiments, the fiber or the MaSp-based fiber is covalently bound to the anti-microbial agent via a linker. In some embodiments, the linker is bound to a tyrosine of the MaSp-based fiber. In some embodiments, the linker is bound to a tyrosine via a diazo group. In some embodiments, the phenol ring of tyrosine undergoes diazotation, so as to form a covalent bond with the linker, thereby forming the anti-microbial agent covalently bound to the MaSp-based fiber.

In some embodiments, the MaSp-based fiber bound to the anti-microbial agent is as represented below:

wherein R comprises the anti-microbial agent as described herein, and represents the MaSp-based fiber. In some embodiments, R comprises a

polymer, such as the antimicrobial polymer (e.g. PGA), or the antimicrobial peptide described herein.

In some embodiments, the linker is 2-(4-aminopehyl)ethylamine. In some embodiments, the MaSp-based fiber bound to the anti-microbial agent via a diazotized 2-(4-aminopehyl)ethylamine. In some embodiments, R comprising the anti-microbial agent is represented by any of the structures provided below:

wherein: X represents a heteroatom; n is an integer being between 0 and 10; dye comprises the photosensitizer; and R comprises an optionally substituted alkyl, an optionally substituted aryl, an optionally substituted heteroaryl, or a combination thereof.

General

As used herein the term “about” refers to +10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the term “stably encapsulated” refers to the ability of the composition to substantially prevent a release of the active ingredient therefrom. As used herein, the term “substantially prevent” is referred to a total amount of the active ingredient removed by the first tape strip and by the second tape strip, as measured by a skin tape test (as described in the Examples section).

In some embodiments, substantially comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, including nay range between. As used herein, the term “bound” refers to a covalent bond (e.g. coordinative bond, a single bond, a double bond, a triple bond, etc.), a non-covalent bond, a physical interaction or a combination thereof. Non-covalent bonds are well-known in the art and include inter alia hydrogen bonds, p-p stacking, Van der Waals interactions, etc.

The word “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

The word “optionally” is used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.

As used herein, the singular form “a” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

Definitions

As used herein, the term “alkyl” describes an aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has between 1 to 10 carbon atoms, and more preferably 1-6 carbon atoms (or C₁-C₆ alkyl). A short alkyl therefore has 20 or less main-chain carbons. The alkyl can be substituted or unsubstituted, as defined herein.

The term “alkyl”, as used herein, also encompasses saturated or unsaturated hydrocarbon, hence this term further encompasses alkenyl and alkynyl. As used herein the term “C₁-C₆ alkyl” including any C₁-C₆ alkyl related compounds, is referred to any linear or branched alkyl chain comprising between 1 and 6, between 1 and 2, between 2 and 3, between 3 and 4, between 4 and 5, between 5 and 6, carbon atoms, including any range therebetween. In some embodiments, C₁-C₆ alkyl comprises any of methyl, ethyl, propyl, butyl, pentyl, iso-pentyl, hexyl, and tert-butyl or any combination thereof. In some embodiments, C₁-C₆ alkyl as described herein further comprises an unsaturated bond, wherein the unsaturated bond is located at 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th) or 6^(th) position of the C₁-C₆ alkyl.

As used herein the term “C₁-C₁₀ alkyl” including any C₁-C₆ alkyl related compounds, is referred to any linear or branched alkyl chain comprising between 1 and 6, between 1 and 2, between 2 and 3, between 3 and 4, between 4 and 5, between 5 and 6, between 6 and 8, between 8 and 10, carbon atoms, including any range therebetween. In some embodiments, C₁-C₁₀ alkyl comprises any of methyl, ethyl, propyl, butyl, pentyl, iso-pentyl, hexyl, nonyl, decyl and tert-butyl or any combination thereof. In some embodiments, C₁-C₁₀ alkyl as described herein further comprises an unsaturated bond, wherein the unsaturated bond is located at 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th), 8^(th), 9^(th) or 10^(th) position of the C₁-C₁₀ alkyl.

The term “alkenyl” describes an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.

The term “alkynyl”, as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.

The term “cycloalkyl” describes an all-carbon monocyclic or fused ring (i.e. rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or unsubstituted, as indicated herein. In some embodiments, the term “cycloalkyl” refers to a C3-C10 cyclic ring. In some embodiments, the term “cycloalkyl” refers to a C3-C10 cyclic ring comprising 1, 2, 3, or 4 heteroatoms (e.g. N, NH, O, or S). (C₃-C₁₀) ring is referred to an optionally substituted C3, C4, C5, C6, C7, C8, C9 or C10 ring. In some embodiments, (C₃-C₁₀) ring comprises optionally substituted cyclopropane, cyclobutene, cyclopentane, cyclohexane, or cycloheptane.

The term “aryl” describes an all-carbon monocyclic or fused-ring polycyclic (i.e. rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. The term “aryl” describes an aromatic (C₆-C₁₂) ring. The aryl group may be substituted or unsubstituted, as indicated herein. As used herein the term “(C₆-C₁₂) ring” is referred to an optionally substituted C6, C7, C8, C9, C10, C11, or C12 aromatic ring. In some embodiments, (C₆-C₁₂) aromatic ring is referred to a bicyclic aryl or bicyclic heteroaryl (e.g. fused ring, spirocyclic ring, and biaryl ring).

As used herein the term “bicyclic heteroaryl” referred to (C₆-C₁₂) a bicyclic heteroaryl ring, wherein bicyclic (C₆-C₁₀) ring is as described herein.

As used herein the term “bicyclic aryl” referred to (C₆-C₁₂) a bicyclic aryl ring, wherein bicyclic (C₆-C₁₂) ring is as described herein.

The term “alkoxy” describes both an O-alkyl and an —O-cycloalkyl group, as defined herein.

The term “aryloxy” describes an —O-aryl, as defined herein.

Each of the alkyl, cycloalkyl and aryl groups in the general formulas herein may be substituted by one or more substituents, whereby each substituent group can independently be, for example, halide, alkyl, alkoxy, cycloalkyl, nitro, amino, hydroxyl, thiol, thioalkoxy, carboxy, amide, aryl and aryloxy, depending on the substituted group and its position in the molecule. Additional substituents are also contemplated.

The term “halide”, “halogen” or “halo” describes fluorine, chlorine, bromine, or iodine.

The term “haloalkyl” describes an alkyl group as defined herein, further substituted by one or more halide(s).

The term “haloalkoxy” describes an alkoxy group as defined herein, further substituted by one or more halide(s).

The term “hydroxyl” or “hydroxy” describes a —OH group.

The term “mercapto” or “thiol” describes a —SH group.

The term “thioalkoxy” describes both an —S-alkyl group, and a —S-cycloalkyl group, as defined herein.

The term “thioaryloxy” describes both an —S-aryl and a —S-heteroaryl group, as defined herein.

The term “amino” describes a —NR′R″ group, with R′ and R″ as described herein.

The term “heterocyclyl” describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen, and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino and the like.

The term “carboxy” or “carboxylate” describes a —C(O)OR′ group, where R′ is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon) or heterocyclyl (bonded through a ring carbon) as defined herein.

The term “carbonyl” describes a —C(O)R′ group, where R′ is as defined hereinabove.

The above-terms also encompass thio-derivatives thereof (thiocarboxy and thiocarbonyl).

The term “thiocarbonyl” describes a —C(S)R′ group, where R′ is as defined hereinabove.

A “thiocarboxy” group describes a —C(S)OR′ group, where R′ is as defined herein.

A “sulfinyl” group describes an —S(O)R′ group, where R′ is as defined herein.

A “sulfonyl” or “sulfonate” group describes an —S(O)₂R′ group, where R′ is as defined herein.

A “carbamyl” or “carbamate” group describes an —OC(O)NR′R″ group, where R′ is as defined herein and R″ is as defined for R′.

A “nitro” group refers to a —NO₂ group.

The term “amide” as used herein encompasses C-amide and N-amide.

The term “C-amide” describes a —C(O)NR′R″ end group or a —C(O)NR′-linking group, as these phrases are defined hereinabove, where R′ and R″ are as defined herein.

The term “N-amide” describes a —NR″C(O)R′ end group or a —NR′C(O)— linking group, as these phrases are defined hereinabove, where R′ and R″ are as defined herein.

The term “carboxylic acid derivative” as used herein encompasses carboxy, amide, carbonyl, anhydride, carbonate ester, and carbamate.

A “cyano” or “nitrile” group refers to a —CN group.

The term “azo” or “diazo” describes an —N═NR′ end group or an —N═N— linking group, as these phrases are defined hereinabove, with R′ as defined hereinabove.

The term “guanidine” describes a —R′NC(N)NR″R′″ end group or a —R′NC(N) NR″-linking group, as these phrases are defined hereinabove, where R′, R″ and R′″ are as defined herein.

As used herein, the term “azide” refers to a —N3 group.

The term “sulfonamide” refers to a —S(O)₂NR′R″ group, with R′ and R″ as defined herein.

The term “phosphonyl” or “phosphonate” describes an —OP(O)—(OR′)₂ group, with R′ as defined hereinabove.

The term “phosphinyl” describes a —PR′R″ group, with R′ and R″ as defined hereinabove.

The term “alkylaryl” describes an alkyl, as defined herein, which substituted by an aryl, as described herein. An exemplary alkylaryl is benzyl.

The term “heteroaryl” describes a monocyclic (e.g. C5-C6 heteroaryl ring) or fused ring (i.e. rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen, and sulfur and, in addition, having a completely conjugated pi-electron system. In some embodiments, the terms “heteroaryl” and “C5-C6 heteroaryl” are used herein interchangeably. Examples, without limitation, of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted by one or more substituents, as described hereinabove. Representative examples are thiadiazol, pyridine, pyrrole, oxazole, indole, purine, and the like.

As used herein, the terms “halo” and “halide”, which are referred to herein interchangeably, describe an atom of a halogen, that is fluorine, chlorine, bromine, or iodine, also referred to herein as fluoride, chloride, bromide, and iodide.

The term “haloalkyl” describes an alkyl group as defined above, further substituted by one or more halide(s).

The term “substituted” or the term “substituent” is referred to 1, 2, 3, 4 or 5 substituents, wherein each substituent is independently selected from (C₀-C₆)alkyl-aryl, (C₀-C₆)alkyl-heteroaryl, (C₀-C₆)alkyl-(C₃-C₅) cycloalkyl, optionally substituted C₃-C₈ heterocyclyl, halogen, —NO₂, —CN, —OH, —CONH₂, —CONR₂, —CNNR₂, —CSNR₂, —CONH—OH, —CONH—NH₂, —NHCOR, —NHCSR, —NHCNR, —NC(═O)OR, —NC(═O)NR, —NC(═S)OR, —NC(═S)NR, —SO₂R, —SOR, —SR, —SO₂OR, —SO₂N(R)₂, —NHNR₂, —NNR, C₁-C₆ haloalkyl, optionally substituted C₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, hydroxy(C₁-C₆ alkyl), hydroxy(C₁-C₆ alkoxy), alkoxy(C₁-C₆ alkyl), alkoxy(C₁-C₆ alkoxy), C₁-C₆ alkyl-NR2, C₁-C₆ alkyl-SR, —CONH(C₁-C₆ alkyl), —CON(C₁-C₆ alkyl)₂, —CO₂H, —CO₂R, —OCOR, —OCOR, —OC(═O)OR, —OC(═O)NR, —OC(═S)OR, —OC(═S)NR, including nay combination thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

Example 1 Derivatized MaSp-Based Fibers

Amination of MaSp Based Fibers has been Performed as Follows:

Aqueous dispersion comprising MaSp based fibers (also used herein as “SVX”) together with an anionic surfactant, was centrifuged and re-dispersed in deionized (DI) water. Furthermore, MaSp based fibers have been substantially dried according to a well-established procedure and re-dispersed in DI water, so as to obtain a MaSp suspension.

Aqueous solution (1-20% w/w) of 2-(4-aminophenyl)ethylamine (APEA) has been prepared, followed by acidification with HCl (1M).

2-10 molar excess of NaNO₂ has been added to the 2-(4-aminophenyl)ethylamine solution and kept at a temperature between 1 and 10° C. for 10-60 minutes, so as to obtain diazotation solution. Then, the diazotation solution was added dropwise to the MaSp suspension while cooling. The resulting mixture has been stirred under completion of the reaction.

Then, the supernatant has been discarded and the remaining derivatized MaSp-based fibers have been extensively washed with DI water, followed by drying of the aminated fibers.

The modification efficiency (yield) has been determined by calculating the amount of unreacted APEA in the reaction mixture. The calculation has been performed by determining the UV absorption of APEA (via UV-spectrophotometry). Up to 60% tyrosine diazotation was observed.

Conjugation of the Aminated MaSp Based Fibers with Polyglutaraldehyde (PGA) has been Performed as Follows:

Preparation of polyglutaraldehyde (average M.W. of between 500 and 2000, or of about 1000 Da):

20 ml of glutaraldehyde aquatic solution 25% was added to K2CO3 1M aquatic solution and heated to 50° C. for 2 hours. Then cooled to R.T. (room temperature, between 20 and 25 deg.C) and pH was set to 7 using HCl 37%. Then it was centrifuged at 7000 rpm for 10 min and the soup was diluted with acetone to 10 times of its volume. The K2CO3 powder was filtered out and acetone was allowed to vaporize. The aquatic PGA solution was lyophilized and kept at −20° C.

Aminated MaSp based fibers have been added to 1-10 ml of polyglutaraldehyde (PGA) solution [at a concentration of 0.01-2 M] in an aqueous buffer. Then, the resulting suspension was cooled to R.T. and the pH was neutralized using HCl (37%). The reaction was kept at 1-10° C. overnight under stirring to result the desired product: SVX-PGA.

Conjugation of the Metal Chelating Group (IDA) to the Polyglutaraldehyde (PGA) has been Performed as Follows:

MaSp based fibers derivatized with PGA was added to sodium iminodiacetate dibasic hydrate (IDA) 0.28M solution buffered with HEPES (0.05M) and left overnight in an ice bath (1-10° C.), and subsequently washed by re-dispersion in HEPES buffer.

The reaction mixture has been centrifuged and the precipitant re-dispersed in 10 ml of an aqueous buffer, followed by centrifugation and extensive washings with DI water. The resulting conjugate (PGA-IDA modified MaSp-based fiber) has been lyophilized.

Conjugation of the Aminated MaSp Based Fibers with Poly(Acrylic Acid) (PAA)

1.84 g of poly(acrylic acid) (Aldrich 306223, Average M.W. of about 3,000,000 Da) were suspended in 0.1M MES (N-morpholino)ethanesulfonic acid) buffered solution over night at R.T. with stirring. Then, aminated MaSp based fibers (prepared as described above) were suspended in MES (0.1M), and the suspension was added to the poly(acrylic acid) suspension and left at stirring at least 4 hours until a uniform suspension was obtained. Then an aqueous solution of 10-30 mg EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide*HCl) in 5 ml DI water was added to the suspension. Followed by addition of an aqueous solution of 40 mg NHS (N-Hydroxysulfosuccinimide sodium salt) in 5 ml DI water. The resulting mixture was left to stir overnight at R.T (room temperature). The reaction mixture was centrifuged and re-suspended in water for several times. The resulting conjugate (PAA modified MaSp-based fiber) has been lyophilized.

The PAA/MaSp fiber molar or w/w ratio can be determined via FTIR spectroscopy by calculating the ratio between peak intensities of a peak at 1695 cm⁻¹ (corresponding to PAA) and a peak at 1620 cm⁻¹ (corresponding to MaSp fiber). The peak intensities are normalized to the concentration, based on a calibration curve.

The inventors successfully synthesized PAA modified MaSp-based fibers, with a w/w ratio between PAA and MaSp fiber ranging from 1 to 200%, and a molar ratio between PAA and MaSp fiber ranging from 5*10⁻⁴ to 5*10⁻².

Conjugation of the Carboxylated MaSp Based Fibers with Poly(Acrylamide) (PAAm)

Carboxylation of MaSp Based Fibers has been Performed as Follows:

Aqueous dispersion comprising MaSp based fibers (also used herein as “SVX”) together with an anionic surfactant, was centrifuged and re-dispersed in deionized (DI) water. Furthermore, MaSp based fibers have been substantially dried according to a well-established procedure and re-dispersed in DI water, so as to obtain a MaSp suspension.

Aqueous solution (1-20% w/w) of 4-aminobenzoic acid or of 4-aminophenylacetic acid has been prepared, followed by acidification with HCl (1M).

2-10 molar excess of NaNO₂ has been added to the 4-aminobenzoic acid or of 4-aminophenylacetic acid solution and kept at a temperature between 1 and 10° C. for 10-60 minutes, so as to obtain diazotation solution. Then, the diazotation solution was added dropwise to the MaSp suspension while cooling. The resulting mixture has been stirred under completion of the reaction.

Then, the supernatant has been discarded and the remaining derivatized MaSp-based fibers have been extensively washed with DI water, followed by drying of the carboxylated fibers, to result the desired product (SVX-COOH).

The modification efficiency (yield) has been determined by calculating the amount of unreacted 4-aminobenzoic acid or of 4-aminophenylacetic acid in the reaction mixture. The calculation has been performed by determining the UV absorption of 4-aminobenzoic acid or of 4-aminophenylacetic acid (via UV-spectrophotometry). Up to 60% tyrosine diazotation was observed.

Conjugation of SVX-COOH with Poly(Acrylamide) (PAAm) Via In-Situ Polymerization

500 mg of acid modified SVX-COOH (prepared as described above) in an aquatic suspension were centrifuged and re-suspended in MES buffer (0.1M).

40 L of DMPA 3-(Dimethylamino)-1-propylamine were added to the suspension. Then, an aqueous solution of 13.3 mg EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide*HCl) in 5 ml DI water was added to the suspension, followed by addition of 20 mg NHS (N-Hydroxysulfosuccinimide sodium salt) in 5 ml DI. The reaction mixture was stirred for 0.5-10 h at R.T, followed by centrifugation and washing with DI water.

Subsequently, 0.1-5 g of acrylamide was dissolved in 1-10 ml of water and added to the SVX-COOH-DMPA suspension and shook firmly. Then, 100 L of ammonium persulfate 10% solution were added to the SVX-COOH-DMPA suspension and the resulting mixture was stirred at R.T overnight, followed by centrifugation and washing with DI water, so as to obtain the desired conjugate via PAAm in-situ polymerization on SVX-COOH.

The inventors successfully synthesized conjugates of the derivatized MaSp-based fiber with various polymers such as PGA, PAA, PVA, PEI, PAAm or a combination thereof (such as PGA-co-PEI). Furthermore, inventors successfully synthesized abovementioned conjugates using a MaSp-based protein having a mutant amino acid sequence (also used herein as “mutant MaSp-based protein”).

It is postulated, that the outstanding porosity (e.g. defined by a BET surface area of at least 10 m²/g) of the derivatized MaSp-based fibers is sequence independent, since both MaSp-based protein and the mutant MaSp-based protein exhibit a highly porous structure.

Example 2 Derivatized MaSp-Based Fibers Doped with a Metal

The inventors successfully synthesized an aminated MaSp-based fiber bound to PGA wherein PGA is further bound to a metal chelating group (iminodiacetate or IDA=IHN(CH)₁CO₂H)₂), synthesized according to the procedure of Example 1. Furthermore, the PGA-IDA modified MaSp-based fiber was successfully doped with Cu (0) deposited via electroless deposition method.

Doping of PGA-IDA Modified MaSp-Based Fiber with Pd:

PGA-IDA modified MaSp-based fiber was dispersed in 0.05M HEPES buffer. Then Palladium (II) acetate aqueous solution (3 mM) was added to the dispersion and the reaction was kept at 1-10° C. overnight under stirring. Then the modified MaSp-based fiber was separated via centrifugation and extensively washed with DI water. Subsequently another portion of Pd(Acetate)₂ was added to the modified MaSp-based fiber and the reaction mixture was stirred for 1-10 hours at R.T.

The reaction mixture has been centrifuged and the precipitant re-dispersed in 10 ml of an aqueous buffer, followed by centrifugation and extensive washings with DI water. The resulting composite (Pd modified MaSp-based fiber) has been lyophilized.

Reduction of Pd to Pd(0):

The reduction mixture based on lactic acid and boron complex was added to Pd modified MaSp-based fiber to induce Pd (II) reduction to achieve Pd (0). The obtained grey fibers washed several times with water to remove all traces of unreacted reduction mixture.

Copper Deposition:

The suspension of Pd (0) modified MaSp-based fiber was treated with standard Cu solution for electroless deposition. During the process the blue color of Cu (II) disappeared, and the fibers turned brown. The obtained Cu (0) doped MaSp-based fibers were washed several times with water.

Alternatively, copper colloids absorbed (non-covalently bound) on or within MaSp based fibers (as illustrated in FIG. 1 ) have been prepared as follows:

Treatment with PEI:

Pristine MaSp-based fibers dispersed in water were mixed with PEI under stirring at 40 deg.C C for 4 hours. The obtained suspension was washed several times with water and dried, resulting in the formation of SVX-PEI composite.

Nucleation with Pd:

Sodium tetrachloropalladate (Na₂PdCl₄) dissolved in water and added to SVX-PEI composite dispersed in water. The mixture stirred for 2 hours to obtain yellow fibers. The obtained suspension of SVX-PEI-Pd (II) was washed with water to remove the excess of unreacted Pd salt.

Subsequently Reduction of Pd to Pd (0) and Copper deposition have been performed as described above, to obtain Cu colloids adsorbed to the SVX-PEI composite. Subsequently, a Nylon thread enriched with 10% of the Cu doped fiber has been prepared and tested for conductivity and anti-microbial activity.

As shown in FIG. 4 , a Nylon thread enriched with 10% of the Cu doped fiber substantially prevented bacterial attachment thereto.

The electrical conductivity of nylon threads enriched with 10% of the Cu (0) doped MaSp-based fibers has been determined, resulting in highly conductive threads (R=0.06 Ω/m). In contrast, the derivatized MaSp-based fiber devoid of metal doping was characterized by a resistivity of more than 10¹⁰ Ω/m.

As shown in FIGS. 2 and 3 , modified MaSp-based fiber and Nylon fibers enriched therewith were successfully coated with palladium and subsequently with copper. In contrary, applying the above-mentioned procedure on “pristine” (i.e. non-enriched) nylon fibers did not result in Cu deposition on top of the pristine fiber. Without being bound to any particular theory, it is postulated that modified MaSp-based fibers facilitate metal (e.g. Pd and/or Cu) deposition.

Example 3 Hair Coloring Compositions

Inventors successfully implemented various derivatized porous MaSp-based fibers in hair coloring compositions (such as the hair coloring compositions described herein). Some of these hair coloring compositions resulted in a uniform and stable hair coating, upon contacting the hair coloring composition with hair (e.g. human hair). Exemplary hair coloring composition which have been successfully implemented for hair coating include aminated MaSp-based fiber (e.g. chemically modified by 4-(2-aminoethyl)aniline; 3-amonipropyltriethoxysilane or by PEI). Additionally, several dyes (cationic and anionic dyes) have been implemented into hair coating, resulting in a colored hair. The colored hair formed upon application of the hair coloring composition described herein, retained its color even upon extensive washings.

Furthermore, the inventors utilized various MaSp-based fibers for the hair coloring compositions. A stable hair coating has been obtained by using aminated MaSp-based protein and aminated mutant MaSp-based protein.

Example 4 Sunscreen Compositions

The inventors successfully synthesized PGA-derivatized MaSp-based fiber modified with the metal oxide chelating agent (succinic acid), as represented by Formula 4.

The modified MaSp-based fiber of Formula 4 (SVX-PGA-SA) has been synthesized by reacting SVX-PGA (prepared according to the procedure of Example 1) with 4-amino salicylic acid as follows:

300 mg of SVX-PGA in water dispersion at pH of 10.5 were mixed with 150 mg (excess) of 4-amino salicylic acid for 1-10 hours at 20-40 degrees C. The color of the reaction mixture changed to orange. Then the reaction mixture has been centrifuged, and the solid SVX-PGA-SA was washed several times to remove any unreacted amino salicylic acid.

Titania particles (particle sized of between 300 and 500 nm) have been complexed by SVX-PGA-SA to result in a composite TiO₂-SVX-PGA-SA as follows:

200 mg of SVX-PGA-SA in water dispersion at pH of 3.5 were mixed with 200 mg Titanium Oxide for 10 hours at 40 degrees C. The color of the reaction mixture changed to deep red. Then the reaction mixture has been centrifuged, and the solid TiO₂-SVX-PGA-SA was washed several times to remove any unreacted materials.

A composite of the invention comprising titanium oxide particles having a particle size of above 300 nm exhibited a significantly improved dispersibility in an aqueous solution and/or organic solution, compared to pristine titania particles. An aqueous dispersion comprising titanium oxide particles bound (complexed) via salicylate bound to PGA-derivatized MaSp-based fiber (e.g. as represented by Formula 4) demonstrated superior stability over a control dispersion comprising non-derivatized MaSp-based fibers. Furthermore, a composite comprising titanium oxide particles bound to salicylate-derivatized MaSp-based fibers demonstrated superior dispersibility (e.g. capable of forming a stable dispersion), wherein a w/w ratio of the titanium oxide particles to the derivatized MaSp-based fibers is about 1:1.

The abovementioned composites have been utilized for reducing UV-exposure (e.g. to the human skin).

Example 5 Thermal Stability of Major Ampullate Spidroin Protein (MaSp)-Based Polymer

By analysis of the differential scanning calorimetry (DSC) curves of the presented spider silk polymers expressed in bacteria (SVX-E), it can be observed that the SVX-E does not present a melting peak. Instead, it showed small glass transition temperature (T_(g)) regions at approximately 220° C. and at approximately 280° C. It could also be observed a degradation peak at approximately 330° C. (FIG. 8 ).

FIG. 8 presents DSC curves of the SVX-E at a temperature increase of 25° C.-280° C. (curve 1), cooling back to 50° C. (curve 2), and new increase to 350° C. (curve 3).

The thermogravimetric analysis (TGA) curves of SVX-E show that at a heating rate of 10° C./min, the weight loss at a temperature bellow 100° C., was only approximately 5% of the absorbed water. It can be observed that a great weight reduction (more than 1%/h) begins at more than 230° C.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. 

1. A derivatized porous major ampullate spidroin protein (MaSp)-based fiber, wherein: said derivatized porous MaSp-based fiber is characterized by a BET surface area of at least 10 m²/g; said derivatized porous MaSp-based fiber comprises a functional moiety covalently bound to a tyrosine of the porous MaSp-based fiber; said functional moiety comprises any one of: amino, carboxy, nitro, sulfonate, carbonyl, ester, anhydride, carbonate ester, carbamate, cyano, hydroxy, a polymer, or any combination thereof.
 2. The derivatized porous MaSp-based fiber of claim 1, wherein a loading of said functional moiety within derivatized porous MaSp-based fiber is between 0.01 μmol/g and 10 mmol/g.
 3. The derivatized porous MaSp-based fiber of claim 1, wherein said functional moiety is covalently bound to a side chain of said tyrosine via a diazo bond, a silyl group, or any combination thereof.
 4. The derivatized porous MaSp-based fiber of claim 1, wherein said polymer is covalently bound to a chelating agent, an antimicrobial agent or any combination thereof; wherein said chelating agent comprises (i) metal chelating group capable of binding a metal or a salt thereof, (ii) a metal oxide chelating group, or both (i) and (ii): optionally wherein a w/w ratio of said polymer to said porous MaSp-based fiber is between 0.001:1 and 5:1.
 5. (canceled)
 6. The derivatized porous MaSp-based fiber of claim 1, wherein said metal chelating group comprises iminodiacetate (IDA), DOTA, NOTA, NODA, EDTA, HBED-CC, including any salt, a derivative, or a combination thereof; and wherein said metal oxide chelating group is selected from salicylic acid, phosphonic acid, hydroxamic acid, malonic acid, pyrogallol, and 5-hydroxy-1,4-naphtoquinone, including any salt, or any combination thereof.
 7. (canceled)
 8. The derivatized porous MaSp-based fiber of claim 1 wherein said polymer is selected from the group consisting of polyglutaraldehyde (PGA), polyvinyl alcohol (PVA), polyacrylate (PAA), polyethyleneimine (PEI), polyacrylamide (PAAm), polylysine, polyaniline, polyurethane, polyamide, polyvinyl chloride, silicon crosspolymer, polyvinyl pyrrolidone or any combination thereof.
 9. (canceled)
 10. The derivatized porous MaSp-based fiber of claim 1, wherein said functional moiety is further bound to a dye or a pigment.
 11. The derivatized porous MaSp-based fiber of claim 1, wherein said MaSp-based fiber is characterized by a degradation temperature (T_(d)) between 280° C. and 350° C. as determined by differential scanning calorimetry (DSC), and by a glass transition temperature (T_(g)) between 200° C. and 250° C., as determined by DSC: optionally wherein said MaSp-based fiber comprises a repetitive region comprising an amino acid sequence set forth in Formula 10: (X₁)_(z)X₂GPGGYGPX₃X₄X₅GPX₆GX₇GGX₈GPGGPGX₉X₁₀; wherein X₁ is, independently, at each instance A or G, Z is an integer between 5 to 30, X₂ is S or G: X₃ is G or E; X₄ is G, S or N: X₅ is Q or Y: X₆ is G or S; X₇ is P or R; X₈ is Y or Q; X₉ is G or S; and X₁₀ is S or G.
 12. (canceled)
 13. A composite comprising the derivatized porous MaSp-based fiber of claim 1 bound to any one of a metal, a salt thereof, and a metal oxide particle or any combination thereof; optionally wherein said metal, the salt thereof, or said metal oxide particle is bound to the derivatized porous MaSp-based fiber via a chelating agent: wherein said chelating agent is covalently bound to said functional moiety.
 14. (canceled)
 15. The composite of claim 13, wherein said chelating agent comprises (i) metal chelating group capable of binding a metal or a salt thereof, (ii) a metal oxide chelating group, or both (i) and (ii); and wherein a molar ratio of said chelating agent to the derivatized porous MaSp-based fiber is between 0.01 and
 1. 16. The composite of claim 13, wherein said metal chelating group comprises iminodiacetate (IDA), DOTA, NOTA, NODA, EDTA, HBED-CC, including any salt, a derivative, or a combination thereof; and wherein said metal oxide chelating group is selected from salicylic acid, phosphonic acid, hydroxamic acid, malonic acid, pyrogallol, and 5-hydroxy-1,4-naphtoquinone, including any salt, or any combination thereof.
 17. (canceled)
 18. The composite of claim 13, wherein said metal oxide particle is selected from titania, zirconia, silica or any combination thereof; and is characterized by a particle size between 10 and 5,000 nm; and wherein a w/w ratio of said derivatized porous MaSp-based fiber to said metal oxide particle within said composite is between 0.01 and
 100. 19. The composite of claim 13, wherein said composite is incorporated into an article selected from a conductive article, an antimicrobial article, and a cosmetic article, including any combination thereof. 20.-28. (canceled)
 29. An article, comprising the derivatized porous MaSp-based fiber of claim
 1. 30. The article of claim 29, wherein said article is a conductive article, an antimicrobial article, or a cosmetic article, including nay combination thereof. 